Methods to control protein heterogeneity

ABSTRACT

The instant invention relates to the field of protein production and in particular to controlled protein heterogeneity compositions and processes for controlling the heterogeneity of proteins expressed in host cells.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. application Ser. No.13/804,220, filed Mar. 14, 2013, which claims priority to U.S.Provisional Application No. 61/696,219, filed on Sep. 2, 2012, thedisclosure of each of which are incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The instant invention relates to the field of protein production and inparticular to processes for controlling and limiting the heterogeneityof proteins expressed in host cells.

BACKGROUND OF THE INVENTION

The production of proteins for biopharmaceutical applications typicallyinvolves the use of cell cultures that are known to produce proteinsexhibiting varying levels of heterogeneity. The basis for suchheterogeneity includes, but is not limited to, the presence of distinctglycosylation substitution patterns. For example, such heterogeneity canbe observed in variations in the fraction of proteins substituted withagalactosyl fucosylated biantennary oligosaccharides NGA2F andNGA2F-GlcNAc and in the fraction of proteins substituted withgalactose-containing fucosylated biantennary oligosaccharides NA1F andNA2F.

Technological advances in recombinant protein production analysis haveprovided unique opportunities for identifying the extent ofheterogeneity exhibited by a particular protein population, particularlyin the context of large-scale production of recombinant proteins.Although such advances have allowed for the robust characterization ofprotein heterogeneity, challenges continue to exist to identify methodsfor producing proteins with desirable heterogeneity. Control of proteinheterogeneity is particularly advantageous in the context of cellculture processes used for commercially produced recombinantbio-therapeutics as such heterogeneity has the potential to impacttherapeutic utility. The instant invention addresses this need byproviding compositions and processes to control protein heterogeneity.

SUMMARY OF THE INVENTION

The present invention is directed to methods for controllingoligosaccharide distribution in a recombinantly-expressed protein sampleand to recombinantly-expressed proteins having defined oligosaccharidedistribution.

In one aspect, the invention is directed to a method for controlling theoligosaccharide distribution of a recombinantly-expressed protein sampleincluding supplementing a cell culture medium used in the recombinantexpression of said protein with a yeast hydrolysate and/or a planthydrolysate. In a related embodiment, the recombinantly-expressedprotein is an antibody or an antigen binding portion thereof. Forexample, the antibody may be an anti-TNFα antibody, such as adalimumab.

In related embodiments, the yeast hydrolysate is selected from the groupconsisting of Bacto TC Yeastolate, HyPep Yeast Extract and UF YeastHydrolysate. In certain embodiments the plant hydrolysate is selectedfrom the group consisting of a soy hydrolysate, a wheat hydrolysate, arice hydrolysate, a cotton seed hydrolysate, a pea hydrolysate, a cornhydrolysate and a potato hydrolysate. For example, the plant hydrolysatemay be selected from the group consisting of BBL Phytone Peptone, HyPep1510, SE50 MAF-UF, UF Soy Hydrolysate, Wheat Peptone E1, HyPep 4601 andProyield WGE80M Wheat.

In certain embodiments, the cell culture medium is supplemented withyeast hydrolysate to achieve a yeast hydrolysate concentration fromabout 2 g/L to about 11 g/L. Alternatively, the cell culture medium issupplemented with yeast hydrolysate to achieve a yeast hydrolysateconcentration of about 2 g/L, 5 g/L or 11 g/L. Alternatively or incombination, the cell culture medium is supplemented with planthydrolysate to achieve a plant hydrolysate concentration from about 2g/L to about 15 g/L. In further related embodiments, the cell culturemedium is supplemented with plant hydrolysate to achieve a planthydrolysate concentration of about 2 g/L, 4 g/L, 7 g/L, 10 g/L or 15g/L.

In certain embodiments of the invention, the cell culture medium issupplemented with yeast hydrolysate and plant hydrolysate to achieve ayeast hydrolysate to plant hydrolysate ratio of about 0.1 to about 4.0.For example, the cell culture medium is supplemented with yeasthydrolysate and plant hydrolysate to achieve a yeast hydrolysate toplant hydrolysate ratio of about 0.25 to about 1.55.

In one embodiment, the recombinantly-expressed protein sample isproduced by a CHO cell line.

In certain embodiments, supplementing the cell culture medium with yeasthydrolysate and/or plant hydrolysate decreases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) present in the protein sample.For example, supplementing the cell culture medium with yeasthydrolysate and/or plant hydrolysate decreases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) by at least about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%.Alternatively or in combination, supplementing the cell culture mediumwith yeast hydrolysate and/or plant hydrolysate decreases the percentageof oligosaccharides NGA2F and (NGA2F-GlcNAc) by about 1%-30%, 2%-25%,5%-20% or 5%-15%. Alternatively or in combination, supplementing thecell culture medium with yeast hydrolysate and/or plant hydrolysatedecreases the percentage of oligosaccharides NGA2F and (NGA2F-GlcNAc) inthe protein sample to about 64%-88%, 70%-88% or 75%-85%.

In certain embodiments, supplementing the cell culture medium with yeasthydrolysate and/or plant hydrolysate increases the percentage ofoligosaccharides NA1F and NA2F present in the protein sample. Forexample, supplementing the cell culture medium with yeast hydrolysateand/or plant hydrolysate increases the percentage of oligosaccharidesNA1F and NA2F by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29% or 30%.

Alternatively or in combination, supplementing the cell culture mediumwith yeast hydrolysate and/or plant hydrolysate increases the percentageof oligosaccharides NA1F and NA2F by about 1%-30%, 2%-25%, 5%-20% or5%-15%. In certain embodiments, supplementing the cell culture mediumwith yeast hydrolysate and/or plant hydrolysate increases the percentageof oligosaccharides NA1F and NA2F in the protein sample to about 8%-31%,10%-25% or 10%-20%.

In certain embodiments, the cell culture medium comprises yeast and/orplant hydrolysate prior to supplementing the medium with the yeasthydrolysate and/or plant hydrolysate. In other embodiments, the cellculture medium is substantially free of yeast and/or plant hydrolysateprior to supplementing the medium with the yeast hydrolysate and/orplant hydrolysate.

In another aspect, the present invention is directed to a method forcontrolling the oligosaccharide distribution of arecombinantly-expressed protein sample including modulating theasparagine and/or glutamine concentration of the cell culture mediumused in the recombinant expression of said protein. In a particularembodiment, the recombinantly-expressed protein sample is an antibody oran antigen binding portion thereof, for example, an anti-TNFα antibodysuch as adalimumab.

In various embodiments, the recombinantly-expressed protein is producedin a CHO cell line.

In certain embodiments, the method includes modulating the concentrationof glutamine and asparagine. For example, the method may includeincreasing the concentration of asparagine. Alternatively or incombination, the method includes increasing the concentration ofglutamine. In a particular embodiment, the concentration of asparagineand/or glutamine in the cell culture medium is modulated to a level ofgreater than about 0.2 g/L. Alternatively, the concentration ofasparagine and/or glutamine in the cell culture medium is modulated to alevel of greater than about 0.4 g/L, 0.6 g/L, 0.8 g/L, 1.0 g/L, 1.2 g/L,1.4 g/L, 1.6 g/L, 1.8 g/L or 2 g/L. In related embodiments, theconcentration of asparagine and/or glutamine in the cell culture mediumis modulated to a level between about 0.4 g/L-1.4 g/L.

In certain embodiments, the cell culture medium includes a hydrolysate,for example, a yeast hydrolysate and/or a plant hydrolysate. In aparticular embodiment, the yeast hydrolysate is selected from the groupconsisting of Bacto TC Yeastolate, HyPep Yeast Extract and UF YeastHydrolysate. In a particular embodiment, the plant hydrolysate isselected from the group consisting of a soy hydrolysate, a wheathydrolysate, a rice hydrolysate, a cotton seed hydrolysate, a peahydrolysate, a corn hydrolysate and a potato hydrolysate. For example,the plant hydrolysate may be selected from the group consisting of BBLPhytone Peptone, HyPep 1510, SE50 MAF-UF, UF Soy Hydrolysate, WheatPeptone E1, HyPep 4601 and Proyield WGE80M Wheat.

In certain embodiments, an increase in the concentration of asparagineand/or glutamine in the cell culture medium increases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) present in the protein sample.For example, an increase in the concentration of asparagine and/orglutamine in the cell culture medium increases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) by at least about 0.1%, 0.2%,0.3%. 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3.0%,3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%. Alternatively orin combination, an increase in the concentration of asparagine and/orglutamine in the cell culture medium increases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) by about 0.5%, 1.0%, 1.5%,2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%,8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 11%, 12%, 13%, 14% or 15%. Alternativelyor in combination, an increase in the concentration of asparagine and/orglutamine in the cell culture medium increases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) by about 0.5%-15%, by about0.5%-10% or by about 4-6%.

In certain embodiments, an increase in the concentration of asparagineand/or glutamine in the cell culture medium decreases the percentage ofoligosaccharides NA1F and NA2F present in the protein sample generatedby the cell line. For example, an increase in the concentration ofasparagine and/or glutamine in the cell culture medium decreases thepercentage of oligosaccharides NA1F and NA2F by at least about 0.5%,1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%,7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 11%, 12%, 13%, 14% or 15%. Ina particular embodiment, an increase in the concentration of asparagineand/or glutamine in the cell culture medium decreases the percentage ofoligosaccharides NA1F and NA2F by about 0.5%-10%, by about 0.5%-6% or byabout 2-5%.

In certain embodiments, the cell culture medium includes asparagineand/or glutamine prior to modulating the concentration of asparagineand/or glutamine. In another embodiment, the cell culture medium issubstantially free of asparagine and/or glutamine prior to modulatingthe concentration of asparagine and/or glutamine.

According to another aspect of the invention, a composition includingthe recombinantly-expressed protein produced by any of the foregoingmethods is provided. For example, the protein may be an anti-TNFαantibody such as adalimumab, or an antigen binding portion thereof.

In one aspect, a pharmaceutical composition including therecombinantly-expressed protein produced by any of the foregoing methodsis provided. For example, the protein may be an anti-TNFα antibody suchas adalimumab, or an antigen binding portion thereof.

In another aspect, the present invention is directed to a compositionincluding N-linked glycosylated adalimumab, such that theoligosaccharides NGA2F and (NGA2F-GlcNAc) are present at about 64%-88%and/or such that the oligosaccharides NA1F and NA2F are present at about8-31%, based on the total amount of oligosaccharides present in thecomposition. For example, the oligosaccharides NGA2F and (NGA2F-GlcNAc)may be present at about 70%-88% or at about 75%-85%. Alternatively or incombination, the oligosaccharides NA1F and NA2F are present at about10%-25% or at about 10%-20%.

In yet another aspect, the present invention is directed to apharmaceutical composition including N-linked glycosylated adalimumaband a pharmaceutically acceptable excipient, such that theoligosaccharides NGA2F and (NGA2F-GlcNAc) are present at about 64%-88%and/or such that the oligosaccharides NA1F and NA2F are present at about8-31%, based on the total amount of oligosaccharides present in thecomposition. For example, the oligosaccharides NGA2F and (NGA2F-GlcNAc)may be present at about 70%-88% or at about 75%-85%. Alternatively or incombination, the oligosaccharides NA1F and NA2F are present at about10%-25% or at about 10%-20%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the effect of yeast, soy or wheat hydrolysate addition toCDM GIA-1 in adalimumab-producing CHO cell line #1 on (a) Culturegrowth, (b) Culture viability and (c) Harvest titer.

FIG. 2 depicts the effect of yeast, soy or wheat hydrolysate addition toCDM GIA-1 in adalimumab-producing CHO cell line #1 on (a) NGA2F and(NGA2F-GlcNac) and (b) NA1F and NA2F.

FIG. 3 depicts the effect of combined supplementation of yeast and soyhydrolysates to CD media from multiple suppliers in adalimumab-producingCHO cell line #1 on (a) Culture growth, (b) Culture viability and (c)Harvest titer.

FIG. 4 depicts the effect of combined supplementation of yeast and soyhydrolysates to CD media from multiple suppliers in adalimumab-producingCHO cell line #1 on (a) NGA2F and (NGA2F-GlcNac) and (b) NA1F and NA2F.

FIG. 5 depicts the effect of supplementing (a) yeast, (b) soy or (c)wheat hydrolysate from multiple vendors to CDM GIA-1 on culture growthin CHO cell line #1.

FIG. 6 depicts the effect of supplementing (a) yeast, (b) soy or (c)wheat hydrolysate from multiple vendors to CDM GIA-1 on cultureviability in CHO cell line #1.

FIG. 7 depicts the effect of supplementing yeast, soy or wheathydrolysate from multiple vendors to CDM GIA-1 on harvest titer in CHOcell line #1.

FIG. 8 depicts the effect of supplementing yeast, soy or wheathydrolysate from multiple vendors to CDM GIA-1 in CHO cell line #1 on(a) NGA2F and (NGA2F-GlcNac) and (b) NA1F and NA2F.

FIG. 9 depicts viable cell density and viability in Example 4:Hydrolysate study #1 using distinct ratios of yeast to soy hydrolysatein adalimumab-producing CHO cell line #1.

FIG. 10 depicts viable cell density and viability in Example 4:Hydrolysate study #2 using distinct ratios of yeast to soy hydrolysatein adalimumab-producing CHO cell line #1.

FIG. 11 depicts the glycosylation profile in Example 4: HydrolysateStudy #1 in adalimumab-producing CHO cell line #1.

FIG. 12 depicts the glycosylation profile in Example 4: HydrolysateStudy #2 in adalimumab-producing CHO cell line #1.

FIG. 13 depicts the effect of supplementation of asparagine and/orglutamine on day 6 to hydrolysate based media in CHO cell line #1 onculture growth (a), culture viability (b) and product titer (c).

FIG. 14 depicts the effect of supplementation of asparagine and/orglutamine on Day 6 to hydrolysate based media in adalimumab-producingCHO cell line #1 on NGA2F and (NGA2F-GlcNac) glycans (a) and on NA1F andNA2F glycans (b).

FIG. 15 depicts the dose dependent effect of supplementation ofasparagine on Day 7 to hydrolysate based media in adalimumab-producingCHO cell line #1 on culture growth (a) and culture viability (b) andproduct titer (c).

FIG. 16 depicts the dose dependent effect of supplementation ofasparagine on Day 7 to hydrolysate based media in adalimumab-producingCHO cell line #1 on NGA2F and (NGA2F-GlcNac) glycans (a) and on NA1F andNA2F glycans (b).

FIG. 17 depicts the dose dependent effect of supplementation ofasparagine on Day 0 to hydrolysate based media in adalimumab-producingCHO cell line #1 on culture growth (a) and culture viability (b) andproduct titer (c).

FIG. 18 depicts the dose dependent effect of supplementation ofasparagine on Day 0 to hydrolysate based media in adalimumab-producingCHO cell line #1 on NGA2F and (NGA2F-GlcNac) glycans (a) and on NA1F andNA2F glycans (b).

FIG. 19 depicts the effect of yeast, soy or wheat hydrolysate additionto CDM Irvine IS CHO-CD in adalimumab-producing CHO cell line #1 on (a)Culture growth, (b) Culture viability and (c) Harvest titer.

FIG. 20 depicts the effect of yeast, soy or wheat hydrolysates additionto CDM Irvine IS CHO-CD in adalimumab-producing CHO cell line #1 onoligosaccharides profile (a) NGA2F and (NGA2F-GlcNac) and (b) NA1F andNA2F.

FIG. 21 depicts the effect of yeast, soy or wheat hydrolysate additionto CDM GIA-1 in adalimumab-producing CHO cell line #2 on (a) Culturegrowth, (b) Culture viability and (c) Harvest titer.

FIG. 22 depicts the effect of yeast, soy or wheat hydrolysate additionto CDM GIA-1 in adalimumab-producing CHO cell line #2 on (a) NGA2F and(NGA2F-GlcNac) and (b) NA1F and NA2F.

FIG. 23 depicts the effect of yeast, soy or wheat hydrolysate additionto CDM GIA-1 in adalimumab-producing CHO cell line #3 on (a) Culturegrowth, (b) Culture viability and (c) Harvest titer.

FIG. 24 depicts the effect of yeast, soy or wheat hydrolysate additionto CDM GIA-1 in adalimumab-producing CHO cell line #3 on (a) NGA2F and(NGA2F-GlcNac) and (b) NA1F and NA2F.

FIG. 25 depicts the effect of yeast, soy or wheat hydrolysate additionto CDM GIA-1 in CHO cell line producing mAb #1 (a) Culture growth, (b)Culture viability and (c) Harvest titer.

FIG. 26 depicts the effect of yeast, soy or wheat hydrolysate additionto CDM GIA-1 in CHO cell line producing mAb #1 on (a) NGA2F and(NGA2F-GlcNac) and (b) NA1F and NA2F.

FIG. 27 depicts the effect of yeast, soy or wheat hydrolysate additionto CDM GIA-1 in CHO cell line producing mAb #2 on (a) Culture growth,(b) Culture viability and (c) Harvest titer.

FIG. 28 depicts the effect of yeast, soy or wheat hydrolysate additionto CDM GIA-1 in CHO cell line producing mAb #2 on (a) NGA2F and(NGA2F-GlcNac) and (b) NA1F and NA2F.

FIG. 29 depicts the effect of combined supplementation of yeast, soyand/or wheat hydrolysates to CDM GIA-1 in adalimumab-producing CHO cellline #1 on (a) Culture growth, (b) Culture viability and (c) Harvesttiter.

FIG. 30 depicts the effect of combined supplementation of yeast, soyand/or wheat hydrolysates to CDM GIA-1 in adalimumab-producing CHO cellline #1 on (a) NGA2F and (NGA2F-GlcNac) and (b) NA1F and NA2F.

FIG. 31 depicts the dose dependent effect of supplementation ofasparagine on Day 6 to CDM GIA-1 in adalimumab-producing CHO cell line#1 on culture growth (a) and culture viability (b) and product titer(c).

FIG. 32 depicts the dose dependent effect of supplementation ofasparagine on Day 6 to CDM GIA-1 in adalimumab-producing CHO cell line#1 on NGA2F and (NGA2F-GlcNac) glycans (a) and on NA1F and NA2F glycans(b).

FIG. 33 depicts the dose dependent effect of supplementation ofasparagine on Day 6 to CDM GIA-1 in adalimumab-producing CHO cell line#2 on culture growth (a) and culture viability (b) and product titer(c).

FIG. 34 depicts the dose dependent effect of supplementation ofasparagine on Day 6 to CDM GIA-1 in adalimumab-producing CHO cell line#2 on NGA2F and (NGA2F-GlcNac) glycans (a) and on NA1F and NA2F glycans(b).

FIG. 35 depicts the dose dependent effect of supplementation ofasparagine during medium preparation to CDM GIA-1 in CHO cell lineproducing mAb #2 on culture growth (a) and culture viability (b) andproduct titer (c).

FIG. 36 depicts the dose dependent effect of supplementation ofasparagine during medium preparation to CDM GIA-1 in CHO cell lineproducing mAb #2 on NGA2F and (NGA2F-GlcNac) glycans (a) and on NA1F andNA2F glycans (b).

FIG. 37 depicts the dose dependent effect of supplementation ofasparagine on Day 5 to CDM GIA-1 in CHO cell line producing mAb #2 onculture growth (a) and culture viability (b) and product titer (c).

FIG. 38 depicts the dose dependent effect of supplementation ofasparagine on Day 5 to CDM GIA-1 in CHO cell line producing mAb #2 onNGA2F and (NGA2F-GlcNac) glycans (a) and on NA1F and NA2F glycans (b).

FIG. 39 depicts the experimental design for Example 1.

FIG. 40 depicts the experimental design for Example 2.

FIG. 41 depicts the experimental design for Example 3.

FIG. 42 depicts the experimental design for Example 6.

FIG. 43 depicts the experimental design for Example 7.

FIG. 44 depicts the experimental design for Example 8.

FIG. 45 depicts the experimental design for Example 9.

FIG. 46 depicts the experimental design for Example 10.

FIG. 47 depicts the experimental design for Example 11 (adaptationstage).

FIG. 48 depicts the experimental design for Example 11 (productionstage).

FIG. 49 depicts the effect of pea hydrolysate addition to CD media GIA-1in adalimumab-producing CHO cell line #1 on Culture growth (a), Cultureviability (b) and Harvest titer (d).

FIG. 50 depicts the effect of pea hydrolysate addition to CD media GIA-1in adalimumab-producing CHO cell line #1 on NGA2F and (NGA2F-GlcNac) (a)and NA1F and NA2F (b).

FIG. 51 depicts the structures of oligosaccharides NGA2F,(NGA2F-GlcNac), NA1F and NA2F.

DETAILED DESCRIPTION

The present invention is directed to compositions and methods forcontrolling protein heterogeneity arising in a population ofrecombinantly expressed proteins. The present invention is predicated,at least in part, on the discovery that controlling the concentration ofhydrolysates, asparagine and/or glutamine in a cell culture mediumallows for control over the oligosaccharide distribution of arecombinantly-expressed protein produced therein.

For example, in one aspect, the present invention provides a method forcontrolling the oligosaccharide distribution of arecombinantly-expressed protein sample by supplementing the cell culturemedium with a yeast hydrolysate and/or a plant hydrolysate. In certainembodiments, supplementing the cell culture medium with yeasthydrolysate and/or plant hydrolysate decreases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) present in the protein sample.Alternatively or in combination, supplementing the cell culture mediumwith yeast hydrolysate and/or plant hydrolysate increases the percentageof oligosaccharides NA1F and NA2F present in the protein sample.

In another aspect, the method for controlling the oligosaccharidedistribution of the recombinantly-expressed protein sample includesmodulating the asparagine and/or glutamine concentration of the cellculture medium used in the recombinant expression of the protein. Incertain embodiments, an increase in the concentration of asparagineand/or glutamine in the cell culture medium increases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) present in the protein sample.Alternatively or in combination, an increase in the concentration ofasparagine and/or glutamine in the cell culture medium decreases thepercentage of oligosaccharides NA1F and NA2F present in the proteinsample generated by the cell line.

In another aspect, the present invention is directed to a proteinproduced by such methods and having the desired oligosaccharidedistribution or pharmaceutical compositions including such protein. Inparticular, the present invention provides a composition comprisingN-linked glycosylated adalimumab, wherein the oligosaccharides NGA2F and(NGA2F-GlcNAc) are present at about 64%-88% and/or wherein theoligosaccharides NA1F and NA2F are present at about 8-31%, based on thetotal amount of oligosaccharides present in the composition.

Consistency in the quality of the glycoproteins is important becauseglycosylation may impact protein solubility, activity and circulatoryhalf-life. (Gawlitzek et al., Effect of Different Cell CultureConditions on the Polypeptide Integrity and N-glycosylation of aRecombinant Model Glycoprotein. Biotechnol. Bioeng. 1995; 46:536-544;and Hayter et al., Glucose-limited Chemostat Culture of Chinese HamsterOvary Cells Producing Recombinant Human Interferon-γ. Biotechnol.Bioeng. 1992; 39:327-335).

DEFINITIONS

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear. However, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms, for example, those characterized by “a” or“an”, shall include pluralities. In this application, the use of “or”means “and/or”, unless stated otherwise. Furthermore, the use of theterm “including,” as well as other forms of the term, such as “includes”and “included”, is not limiting. Also, terms such as “element” or“component” encompass both elements and components comprising one unitand elements and components that comprise more than one unit unlessspecifically stated otherwise.

The term “protein of interest”, as used herein refers to a targetprotein, production and controlled glycosylation of which is desired. Invarious embodiments, the protein of interest is an antibody or anantigen-binding fragment thereof, a soluble protein, a membrane protein,a structural protein, a ribosomal protein, an enzyme, a zymogen, a cellsurface receptor protein, a transcription regulatory protein, atranslation regulatory protein, a chromatin protein, a hormone, a cellcycle regulatory protein, a G protein, a neuroactive peptide, animmunoregulatory protein, a blood component protein, an ion gateprotein, a heat shock protein, an antibiotic resistance protein, afunctional fragment of any of the preceding proteins, anepitope-containing fragment of any of the preceding proteins andcombinations thereof. In a particular embodiment, the protein ofinterest is a monomer.

In a particular embodiment, the protein of interest is an antibody or anantigen binding portion thereof. The term “antibody” includes animmunoglobulin molecule comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region(CH). The heavy chain constant region is comprised of three domains:CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as LCVR or VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH and VL regions can be further subdivided into regionsof hypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term “antibody”, as usedherein, also includes alternative antibody and antibody-like structures,such as, but not limited to, dual variable domain antibodies (DVD-Ig).

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., hIL-12, hTNFα or hIL-18). It hasbeen shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment comprisingthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment comprising the VH and CH1 domains;(iv) a Fv fragment comprising the VL and VH domains of a single arm ofan antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546,the entire teaching of which is incorporated herein by reference), whichcomprises a VH domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883, the entire teachings of which areincorporated herein by reference). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123, theentire teachings of which are incorporated herein by reference). Stillfurther, an antibody may be part of a larger immunoadhesion molecule,formed by covalent or non-covalent association of the antibody with oneor more other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101, the entire teaching of which isincorporated herein by reference) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058, the entire teaching of which is incorporatedherein by reference). Antibody portions, such as Fab and F(ab′)2fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein. In one aspect, the antigen binding portions arecomplete domains or pairs of complete domains.

The term “human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), e.g., in the CDRs and in particular CDR3. Themutations can be introduced using the “selective mutagenesis approach.”The human antibody can have at least one position replaced with an aminoacid residue, e.g., an activity enhancing amino acid residue which isnot encoded by the human germline immunoglobulin sequence. The humanantibody can have up to twenty positions replaced with amino acidresidues which are not part of the human germline immunoglobulinsequence. In other embodiments, up to ten, up to five, up to three or upto two positions are replaced. In one embodiment, these replacements arewithin the CDR regions. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes (see,e.g., Taylor, L. D. et al. (1992) Nucl. Acids Res. 20:6287-6295, theentire teaching of which is incorporated herein by reference) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences(see, Kabat, E. A. et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.In certain embodiments, however, such recombinant antibodies are theresult of selective mutagenesis approach or back-mutation or both.

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds hTNFα is substantially free ofantibodies that specifically bind antigens other than hTNFα). Anisolated antibody that specifically binds hTNFα may bind TNFα moleculesfrom other species. Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals. A suitable anti-TNFαantibody is Adalimumab (AbbVie, Illinois USA).

As used herein, the term “adalimumab,” also known by its trade nameHUMIRA® (AbbVie, Illinois, USA) refers to a human IgG1 antibody thatbinds human tumor necrosis factor α (TNFα). In general, the heavy chainconstant domain 2 (CH2) of the adalimumab IgG-Fc region is glycosylatedthrough covalent attachment of oligosaccharide at asparagine 297(Asn-297). The light chain variable region of adalimumab is providedherein as SEQ ID NO:1 and the heavy chain variable region of adalimumabis provided herein as SEQ ID NO:2. Adalimumab comprises a light chainvariable region comprising a CDR1 of SEQ ID NO:7, a CDR2 of SEQ ID NO:5and a CDR3 of SEQ ID NO:3. Adalimumab comprises a heavy chain variableregion comprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID NO:6 and CDR3of SEQ ID NO:4. The nucleic acid sequence of the light chain variableregion is set forth in SEQ ID NO:9. The nucleic acid sequence of theheavy chain variable region is set forth in SEQ ID NO:10. The fulllength amino acid sequence of the light chain is set forth as SEQ IDNO:11 and the full length amino acid sequence of the heavy chain is setforth as SEQ ID NO:12. Adalimumab is described in U.S. Pat. Nos.6,090,382; 6,258,562; 6,509,015; 7,223,394; 7,541,031; 7,588,761;7,863,426; 7,919,264; 8,197,813; 8,206,714; 8,216,583; 8,420,081;8,092,998; 8,093,045; 8,187,836; 8,372,400; 8,034,906; 8,436,149;8,231,876; 8,414,894; 8,372,401, the entire contents of each which areexpressly incorporated herein by reference in their entireties.Adalimumab is also described in the “Highlights of PrescribingInformation” for HUMIRA® (adalimumab) Injection (Revised January 2008)the contents of which are hereby incorporated herein by reference.

In one embodiment, adalimumab dissociates from human TNFα with a Kd of1×10⁻⁸ M or less and a Koff rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance and neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC50 of 1×10⁻⁷ Mor less. In another embodiment, adalimumab dissociates from human TNFαwith a Koff of 5×10⁻⁴ S⁻¹ or less or even more preferably, with a Koffof 1×10⁻⁴ s⁻¹ or less. In still another embodiment, adalimumabneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC50 of 1×10⁻⁸ M or less, an IC50 of 1×10⁻⁹ M or less or an IC50of 1×10⁻¹⁰ M or less. The term “Koff”, as used herein, is intended torefer to the off rate constant for dissociation of an antibody from theantibody/antigen complex.

As used herein, the term “recombinant host cell” (or simply “host cell”)refers to a cell into which a recombinant expression vector has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell but to the progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein. Incertain embodiments, the host cell is employed in the context of a cellculture.

As used herein, the term “cell culture” refers to methods and techniquesemployed to generate and maintain a population of host cells capable ofproducing a recombinant protein of interest, as well as the methods andtechniques for optimizing the production and collection of the proteinof interest. For example, once an expression vector has beenincorporated into an appropriate host, the host can be maintained underconditions suitable for high level expression of the relevant nucleotidecoding sequences and the collection and purification of the desiredrecombinant protein. Mammalian cells are preferred for expression andproduction of the recombinant of the present invention; however othereukaryotic cell types can also be employed in the context of the instantinvention. See, e.g., Winnacker, From Genes to Clones, VCH Publishers,N.Y., N.Y. (1987). Suitable mammalian host cells for expressingrecombinant proteins according to the invention include Chinese HamsterOvary (CHO cells) (including dhfr-CHO cells, described in Urlaub andChasin, (1980) PNAS USA 77:4216-4220, used with a DHFR selectablemarker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol.159:601-621, the entire teachings of which are incorporated herein byreference), NS0 myeloma cells, COS cells and SP2 cells. Other,non-limiting, examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2), the entire teachings of which are incorporated herein byreference.

The term “cell culture medium” as used herein refers to a combination ofelements in which cells are cultured and which provide nutrients for thegrowth of the cells. A cell culture medium typically contains a mixtureof defined nutrients dissolved in a buffered physiological salinesolution. At a basic level, a cell culture medium contain salts, aminoacids, sugar, vitamins and other organic nutrients. Such medium may beused as a starting point for the addition of various supplements, e.g.,serum (such as fetal bovine serum) and antibiotics to generate acomplete growth medium.

As used herein a “recombinant expression vector” can be any suitablerecombinant expression vector and can be used to transform or transfectany suitable host. For example, one of ordinary skill in the art wouldappreciate that transformation or transfection is a process by whichexogenous nucleic acid such as DNA is introduced into a cell wherein thetransformation or transfection process involves contacting the cell withthe exogenous nucleic acid such as the recombinant expression vector asdescribed herein. Non-limiting examples of such expression vectors arethe pUC series of vectors (Fermentas Life Sciences), the pBluescriptseries of vectors (Stratagene, LaJolla, Calif.), the pET series ofvectors (Novagen, Madison, Wis.), the pGEX series of vectors (PharmaciaBiotech, Uppsala, Sweden) and the pEX series vectors (Clontech, PaloAlto, Calif.).

As used herein, the term “recombinant protein” refers to a proteinproduced as the result of the transcription and translation of a genecarried on a recombinant expression vector that has been introduced intoa host cell. In certain embodiments the recombinant protein is anantibody or an antigen binding portion thereof.

As used herein, the term “glycosylation” refers to the addition of acarbohydrate to an amino acid. Such addition commonly, although notexclusively, occurs via a nitrogen of asparagine or arginine (“N-linked”glycosylation) or to the hydroxy oxygen of serine, threonine, tyrosine,hydroxylysine or hydroxyproline side-chains (“O-linked” glycosylation).In eukaryotes, N-linked glycosylation occurs on the asparagine of theconsensus sequence Asn-Xaa-Ser/Thr, in which Xaa is any amino acidexcept proline (Komfeld et al., Ann Rev Biochem 54: 631-664 (1985);Kukuruzinska et al, Proc. Natl. Acad. Sci. USA 84: 2145-2149 (1987);Herscovics et al, FASEB J. 7:540-550 (1993); and Orlean, SaccharomycesVol. 3 (1996)). O-linked glycosylation also takes place at serine orthreonine residues (Tanner et al., Biochim. Biophys. Acta. 906: 81-91(1987); and Hounsell et al, Glycoconj. J. 13: 19-26 (1996)). However,other glycosylation patterns can be formed, e.g., by linkingglycosylphosphatidyl-inositol to the carboxyl-terminal carboxyl group ofa protein.

In particular embodiments, proteins of the invention are glycosylated bythe addition of NGA2F and (NGA2F-GlcNac) oligosaccharides. As usedherein, the term “NGA2F” refers to an oligosaccharide having theN-linked glycan common core pentasaccharide Man3GlcNAc2, furthercontaining an N-acetylglucosamine linked to each one of the two branchedmannose residues of the core and also containing a fucose linked to theGlcNAc residue proximal to the asparagine residue of the N-linkedglycosylated protein. The term “NGA2F-GlcNAc” as used herein is anoligosaccharide having the N-linked glycan common core pentasaccharideMan3GlcNAc2, further containing one N-acetylglucosamine linked one ofthe two branched mannose residues of the core and also containing afucose linked to the GlcNAc residue proximal to the asparagine residueof the N-linked glycosylated protein. The structures of each of NGA2Fand (NGA2F-GlcNac) are set forth in FIG. 51.

Alternatively or in combination, proteins of the invention areglycosylated by the addition of NA2F and NA1F oligosaccharides. The term“NA2F” as used herein is an oligosaccharide having the N-linked glycancommon core pentasaccharide Man3GlcNAc2, further containing: anN-acetylglucosamine linked to each one of the two branched mannoseresidues of the core, a fucose linked to the GlcNAc residue proximal tothe asparagine residue of the N-linked glycosylated protein and agalactose linked to each N-acetylglucosamine linked to a branchedmannose. The term “NA1F” as used herein is an oligosaccharide having theN-linked glycan common core pentasaccharide Man3GlcNAc2, furthercontaining: an N-acetylglucosamine linked to each one of the twobranched mannose residue of the core, a fucose linked to the GlcNAcresidue proximal to the asparagine residue of the N-linked glycosylatedprotein and a galactose linked to one of the two N-acetylglucosamineresidues linked to a branched mannose. The structures of each of NA2Fand NA1F are set forth in FIG. 51.

As used herein, the term “oligosaccharide distribution” as used hereinrefers to the relative amounts of oligosaccharides found in a sample ofglycosylated proteins. For example, oligosaccharide distribution mayrefer to the relative amounts of N-linked oligosaccharides, i.e., NGA2Fand NGA2F-GlcNac oligosaccharides and NA2F and NA1F oligosaccharidesfound in a sample. According to the methods of the present invention,the oligosaccharide distribution can be controlled, for example, bysupplementing cell culture medium with hydrolysates or by modulating theasparagine and/or glutamine concentration. As used herein, the term“controlled protein heterogeneity” refers to a composition derived fromthe methods of the present invention having a desired oligosaccharidedistribution, for example, a desired ratio of N-linked oligosaccharidesNGA2F and NGA2F-GlcNac oligosaccharides as compared to NA2F and NA1Foligosaccharides.

The term “control”, as used herein, is intended to refer to bothlimitation as well as to modulation. For example, in certainembodiments, the instant invention provides methods for controllingdiversity that decrease the diversity of certain characteristics ofprotein populations and, in particular, glycosylation patterns. Suchdecreases in diversity can occur by: (1) promotion of a desiredcharacteristic, e.g., a favorable glycosylation pattern; (2) inhibitionof an unwanted characteristic, e.g., a disfavored glycosylation pattern;or (3) a combination of the foregoing. As used herein, the term“control” also embraces contexts where heterogeneity is modulated, i.e.,shifted, from one diverse population to a second population of equal oreven greater diversity, where the second population exhibits a distinctprofile of the characteristic of interest. For example, in certainembodiments, the methods of the instant invention can be used tomodulate the types of oligosaccharide substitutions present on proteinsfrom a first population of substitutions to a second equally diverse,but distinct, population of substitutions.

In certain embodiments, the heterogeneity corresponds to theglycosylation state of individual members of a population of proteins.In certain embodiments, control is exerted over the type ofglycosylation substitutions present on individual members of apopulation of proteins. In certain embodiments, control is exerted overthe extent of glycosylation substitutions present on individual membersof a population of proteins. In certain embodiments, control is exertedover both the type and extent of glycosylation substitutions present onindividual members of a population of proteins. In certain embodiments,such control results in a decrease in the amount of NGA2F andNGA2F-GlcNac oligosaccharides and/or an increase in the amount of NA1Fand NA2F oligosaccharides linked to the protein of interest. In certainembodiments, such control results in an increase in the amount of NGA2Fand NGA2F-GlcNac oligosaccharides and/or a decrease in the amount ofNA1F and NA2F oligosaccharides linked to the protein of interest.

In certain embodiments, control over protein glycosylation heterogeneityis exerted by employing specific hydrolysates during production of theprotein of interest, for example, but not by way of limitation, in cellculture media supplemented with hydrolysates. In certain embodiments,control over protein glycosylation heterogeneity is exerted bymaintaining certain yeastolate to phytone ratios during production ofthe protein of interest. In certain embodiments, control over proteinglycosylation heterogeneity is exerted by the addition of asparagineand/or glutamine during the production of the protein of interest.

The term “about”, as used herein, is intended to refer to ranges ofapproximately 10-20% greater than or less than the referenced value. Incertain circumstances, one of skill in the art will recognize that, dueto the nature of the referenced value, the term “about” can mean more orless than a 10-20% deviation from that value.

Control of Protein Heterogeneity

Supplementation of CD Media with Yeast and/or Plant Hydrolysates

The experiments disclosed herein demonstrate that, in certainembodiments, supplementation of CD cell culture media with yeast and/orplant hydrolysates can modulate product quality of a mAb by, in certainembodiments, decreasing the NGA2F and NGA2F-GlcNac oligosaccharides and,in certain embodiments, increasing the NA1F and NA2F oligosaccharides.These results were achieved in multiple CD media available from multiplevendors (Life Sciences Gibco, HyClone and Irvine Scientific), usingyeast and/or plant hydrolysates (for example, but not by way oflimitation, soy, wheat, rice, cotton seed, pea, corn and potato) frommultiple vendors (BD Biosciences organotechnie, Sheffield/KerryBiosciences, Irvine Scientific and DMV International). In experimentswhere yeast or plant hydrolysates were added individually, adose-dependent effect in the extent of reduction of NGA2F andNGA2F-GlcNac oligosaccharides (and a corresponding increase in the NA1Fand NA2F oligosaccharides) with increasing yeast or plant hydrolysatesconcentration in culture CD media was observed. For example, but not byway of limitation, yeast hydrolysates can be used to supplement a CDcell culture media at concentrations ranging from about 1 g/L to about25 g/L, about 1 g/L to about 15 g/L or about 2 g/L to about 11 g/L toachieve the desired reduction of NGA2F and NGA2F-GlcNac oligosaccharidesand a corresponding increase in the NA1F and NA2F oligosaccharides. Incertain non-limiting embodiments, yeast hydrolysates can be used tosupplement a CD cell culture media at concentrations of about 2 g/L,about 5 g/L or about 11 g/L. In certain non-limiting embodiments, planthydrolysates can be used to supplement a CD cell culture media atconcentrations ranging from about 1 g/L to about 30 g/L, about 1 g/L toabout 20 g/L or about 2 g/L to about 15 g/L to achieve the desiredreduction of NGA2F and NGA2F-GlcNac oligosaccharides and a correspondingincrease in the NA1F and NA2F oligosaccharides. In certain non-limitingembodiments, plant hydrolysates can be used to supplement a CD cellculture media at concentrations of about 2 g/L, about 4 g/L, about 7g/L, about 10 g/L or about 15 g/L.

In a particular embodiment, the plant hydrolysate may be a soyhydrolysate. In certain non-limiting embodiments, soy hydrolysates canbe used to supplement a cell culture media at concentrations rangingfrom about 1 g/L to about 30 g/L, about 1 g/L to about 20 g/L or about 2g/L to about 15 g/L to achieve the desired reduction of NGA2F andNGA2F-GlcNac oligosaccharides and a corresponding increase in the NA1Fand NA2F oligosaccharides.

In another embodiment, the plant hydrolysate may be a pea hydrolysate.In certain non-limiting embodiments, pea hydrolysates can be used tosupplement a cell culture media at concentrations ranging from about 1g/L to about 25 g/L, about 1 g/L to about 15 g/L or about 2 g/L to about10 g/L to achieve the desired reduction of NGA2F and NGA2F-GlcNacoligosaccharides and a corresponding increase in the NA1F and NA2Foligosaccharides.

In a particular embodiment, the plant hydrolysate may be a wheathydrolysate. In certain non-limiting embodiments, wheat hydrolysates canbe used to supplement a cell culture media at concentrations rangingfrom about 1 g/L to about 30 g/L, about 1 g/L to about 20 g/L or about 2g/L to about 15 g/L to achieve the desired reduction of NGA2F andNGA2F-GlcNac oligosaccharides and a corresponding increase in the NA1Fand NA2F oligosaccharides.

In certain embodiments, a combination of plant hydrolysates may be used,for example, at least two of soy, pea and wheat hydrolysates. In aparticular embodiment, a combination of each of soy, pea and wheathydrolysates may be used.

In certain embodiments, the concentration of yeast and/or planthydrolysates is maintained in such a manner as to reduce the NGA2F andNGA2F-GlcNac sum in a protein or antibody sample by about 1%, 1.2%,1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100% and ranges within one or more of the preceding. In certainembodiments, the concentration of yeast and/or plant hydrolysates ismaintained in such a manner as to increase the NA1F and NA2F sum in aprotein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%,3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% and ranges withinone or more of the preceding.

In particular embodiments, the cell culture medium includes yeasthydrolysate prior to supplementing the medium as set forth herein. Inanother embodiment, the cell culture medium is substantially free ofyeast hydrolysate prior to supplementing the medium as set forth herein.

In particular embodiments, the cell culture medium includes planthydrolysate prior to supplementing the medium as set forth herein. Inanother embodiment, the cell culture medium is substantially free ofplant hydrolysate prior to supplementing the medium as set forth herein.

In other embodiments, the cell culture medium includes both plant andyeast hydrolysate prior to modulating the concentration of each as setforth herein. In another embodiment, the cell culture medium issubstantially free of both plant and yeast hydrolysate prior tosupplementing the medium as set forth herein.

In certain embodiments, control over the glycosylation distribution ofproteins produced by cell culture can be exerted by maintaining theappropriate yeast hydrolysate concentration in the cell cultureexpressing the protein of interest as described herein. Specific cultureconditions can be used in various cultivation methods including, but notlimited to, batch, fed-batch, chemostat and perfusion and with variouscell culture equipment including, but not limited to, shake flasks withor without suitable agitation, spinner flasks, stirred bioreactors,airlift bioreactors, membrane bioreactors, reactors with cells retainedon a solid support or immobilized/entrapped as in microporous beads andany other configuration appropriate for optimal growth and productivityof the desired cell line.

Modulating Yeast to Plant Hydrolysate Ratio in Cell Culture Medium

According to the present invention the relative amounts of yeast andplant hydrolysates may be modulated to achieve desirable oligosaccharidedistribution. For example, but not by way of limitation, by adjustingthe concentration ratio of these two hydrolysates, yeast and soy(phytone), within the range of about 0.1 to about 4.0, about 0.2 toabout 2.5 or about 0.25 to about 1.55, the resultant oligosaccharidedistribution can be modified. As outlined in Example 1, non-limitingembodiments of the present invention include supplements comprising 100%yeast hydrolysate as well as those that are 100% plant hydrolysate.Thus, this disclosure provides a means to modulate glycosylationvariations introduced by process inputs, such as raw materials and othervariability inherent in dynamic manufacturing operations. Ultimately,the disclosure enables in-process control of protein glycosylation withrespect to desired product specifications.

In certain embodiments, the ratio of these two hydrolysates, yeast andsoy (phytone), is maintained in such a manner as to reduce the NGA2F andNGA2F-GlcNac sum in a protein or antibody sample by about 1%, 1.2%,1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100% and ranges within one or more of the preceding. In certainembodiments, the ratio of these two hydrolysates, yeast and soy(phytone), is maintained in such a manner as to increase the NA1F andNA2F sum in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%,2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%and ranges within one or more of the preceding.

In particular embodiments, the cell culture medium includes yeasthydrolysate and plant hydrolysate prior to adjusting the ratio as setforth herein. In another embodiment, the cell culture medium issubstantially free of yeast and/or plant hydrolysate prior tosupplementing the medium to achieve the desired ratio as set forthherein.

In certain embodiments, control over the glycosylation distribution ofprotein produced by cell culture can be exerted by maintaining theappropriate yeast to plant hydrolysate ratio in the cell cultureexpressing the protein of interest as described herein. Specific cultureconditions can be used in various cultivation methods including, but notlimited to, batch, fed-batch, chemostat and perfusion and with variouscell culture equipment including, but not limited to, shake flasks withor without suitable agitation, spinner flasks, stirred bioreactors,airlift bioreactors, membrane bioreactors, reactors with cells retainedon a solid support or immobilized/entrapped as in microporous beads andany other configuration appropriate for optimal growth and productivityof the desired cell line

Supplementation with Asparagine and/or Glutamine

According to the present invention the concentration of asparagineand/or glutamine may be modified to achieve desirable oligosaccharidedistribution. For example, but not by way of limitation, by adjustingthe concentration of one or both of these two amino acids, the resultantoligosaccharide distribution can be modified. Thus, this disclosureprovides a means to modulate glycosylation variations introduced byprocess inputs, such as raw materials and other variability inherent indynamic manufacturing operations. Ultimately, the disclosure enablesin-process control of protein glycosylation with respect to desiredproduct specifications.

The experiments disclosed herein demonstrate that, in certainembodiments, supplementation of cell culture media with asparagineand/or glutamine can modulate product quality of a mAb by, in certainembodiments, increasing the NGA2F and NGA2F-GlcNac and, in certainembodiments, decreasing the NA1F and NA2F oligosaccharides. For example,but not by way of limitation, the percentage of NGA2F and NGA2F-GlcNaccan be increased by 2-4% and the percentage of NA1F and NA2F wasdecreased by 2-5% when 0.4 to 1.6 g/L asparagine is added on either day0 or days 6 or 7, as outlined in Example 5, below. Similarly, additionof 0.4 g/L glutamine, to the culture run described in Example 5, below,increased the percentage of NGA2F and NGA2F-GlcNac by 1% and lowered thepercentage of NA1F and NA2F by 1%. Finally, adding both asparagine andglutamine (0.4 g/L of each), to the cell culture run described inExample 5, below, increased the percentage of NGA2F and NGA2F-GlcNac by3% and decreased the percentage of NA1F and NA2F by 4%. In addition, thecell growth profile is the same when 0.8 and 1.6 g/L of asparagine wasadded, but a dose dependent effect on oligosaccharide distribution wasobserved, indicating that the effect on oligosaccharide distribution wasdue to the addition of asparagine and not the increased maximum viablecell density or delayed drop in viability.

In certain embodiments, about 0.1 to about 4 g/L, about 0.2 to about 3g/L or about 0.4 to about 2 g/L of asparagine and/or glutamine is addedto the cell culture medium. In a particular embodiment, about 0.1 toabout 4 g/L, about 0.2 to about 3 g/L or about 0.4 to about 2 g/L ofasparagine is added to the cell culture medium. In a particularembodiment, about 0.1 to about 4 g/L, about 0.2 to about 3 g/L or about0.4 to about 2 g/L of glutamine is added to the cell culture medium.

In certain embodiments, the total amount of asparagine and/or glutaminein the cell culture media will range from about 0 mM to about 40 mM orabout 0 mM to about 26 mM. In certain embodiments, for example, thoseembodiments where a hydrolysate media is employed, the range ofasparagine in the cell culture media will range from about 1 mM to about15 mM. In certain embodiments, for example, but not limited to, thoseembodiments where GIA1 media is employed, the range of asparagine in thecell culture media will range from about 12 mM to about 26 mM.

In certain embodiments, the concentration of asparagine and/or glutamineis maintained in such a manner as to reduce the NA1F and NA2F sum in aprotein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%,3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% and ranges withinone or more of the preceding. In certain embodiments, the concentrationof asparagine and/or glutamine is maintained in such a manner as toincrease the NGA2F and NGA2F-GlcNac sum in a protein or antibody sampleby about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100% and ranges within one or more of thepreceding.

In particular embodiments, the cell culture medium includes asparagineprior to modulating the concentration thereof as set forth herein. Inanother embodiment, the cell culture medium is substantially free ofasparagine prior to modulating the concentration thereof as set forthherein.

In particular embodiments, the cell culture medium includes glutamineprior to modulating the concentration thereof as set forth herein. Inanother embodiment, the cell culture medium is substantially free ofglutamine prior to modulating the concentration thereof as set forthherein.

In other embodiments, the cell culture medium includes both asparagineand glutamine prior to modulating the concentration of each as set forthherein. In another embodiment, the cell culture medium is substantiallyfree of both asparagine and glutamine prior to modulating theconcentration thereof as set forth herein.

In certain embodiments, control over the glycosylation distribution ofprotein produced by cell culture can be exerted by maintaining theappropriate asparagine and/or glutamine concentration in the cellculture expressing the protein of interest as described herein. Specificculture conditions can be used in various cultivation methods including,but not limited to, batch, fed-batch, chemostat and perfusion and withvarious cell culture equipment including, but not limited to, shakeflasks with or without suitable agitation, spinner flasks, stirredbioreactors, airlift bioreactors, membrane bioreactors, reactors withcells retained on a solid support or immobilized/entrapped as inmicroporous beads and any other configuration appropriate for optimalgrowth and productivity of the desired cell line.

Assay for Analyzing Oligosaccharide Distribution

Oligosaccharide distribution can be assayed by obtaining a profile ofthe various oligosaccharides present in a sample of the protein ofinterest. By way of example, the first step in the profiling of glycanscan involve releasing the glycan from the protein using enzymaticdigestion with a N-glycanase, e.g. PNGase F or PNGase A. (Varki et al.Essentials of Glycobiology, 2^(nd) Ed. Publisher Cold Spring Harbor,Chapter 47, 2009). N-Glycanase cleaves the GlcNAc-Asn bond, releasingthe N-glycan and converting asparagine into aspartate. Upon release, thefree reducing end of each glycan can be labeled by reductive aminationwith a fluorescent tag such as 2-aminopyridine (2-AP), 2-aminobenzamide(2-AB), 2,6-diaminopyridine (DAP), or biotinylated 2,6-diaminopyridine(BAP). The resulting labeled mixture of glycans can be separated bynormal-phase HPLC (NP-HPLC) and detected by a fluorescence detector. Thefluorescent tag also allows quantitation of the glycan. Thechromatographic profile of the mixture is compared with that of wellcharacterized oligosaccharides standards. Such comparison makes itpossible to estimate the number, relative quantities, and types ofglycans present in a glycoprotein. Individual fractions may be furtheranalyzed by MS or NMR. Sequential treatments with specificexoglycosidases followed by labeling at the reducing end may also beused to obtain structural information of the glycans. A shift in glycanHPLC elution indicates susceptibility to the specific glycosidase.Alternatively or in combination, the oligosaccharide distribution of aprotein sample can be assayed as set forth in the Examples (see, forexample, the materials and methods provided in Example 1.1).

Antibody Generation

Antibodies to be recombinantly produced by the methods of the presentinvention can be generated by a variety of techniques, includingimmunization of an animal with the antigen of interest followed byconventional monoclonal antibody methodologies e.g., the standardsomatic cell hybridization technique of Kohler and Milstein (1975)Nature 256: 495. Although somatic cell hybridization procedures arepreferred, in principle, other techniques for producing monoclonalantibody can be employed e.g., viral or oncogenic transformation of Blymphocytes.

In certain embodiments, the animal system for preparing hybridomas isthe murine system. Hybridoma production is a well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

An antibody can be, in certain embodiments, a human, a chimeric or ahumanized antibody. Humanized antibodies of the present disclosure canbe prepared based on the sequence of a non-human monoclonal antibodyprepared as described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the non-human hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,murine CDR regions can be inserted into a human framework using methodsknown in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen etal.).

Human monoclonal antibodies can be generated using transgenic ortranschromosomic mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice referred to herein as the HuMAb Mouse® (Medarex, Inc.), KMMouse® (Medarex, Inc.) and XenoMouse® (Amgen).

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseantibodies of the disclosure. For example, mice carrying both a humanheavy chain transchromosome and a human light chain tranchromosome,referred to as “TC mice” can be used; such mice are described inTomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727.Furthermore, cows carrying human heavy and light chain transchromosomeshave been described in the art (e.g., Kuroiwa et al. (2002) NatureBiotechnology 20:889-894 and PCT application No. WO 2002/092812) and canbe used to raise the antibodies of this disclosure.

In certain embodiments, the antibodies of this disclosure arerecombinant human antibodies, which can be isolated by screening of arecombinant combinatorial antibody library, e.g., a scFv phage displaylibrary, prepared using human VL and VH cDNAs prepared from mRNA derivedfrom human lymphocytes. Methodologies for preparing and screening suchdisplay libraries (e.g., the Pharmacia Recombinant Phage AntibodySystem, catalog no. 27-9400-01; and the Stratagene SurfZAP™ phagedisplay kit, catalog no. 240612, the entire teachings of which areincorporated herein), examples of methods and reagents particularlyamenable for use in generating and screening antibody display librariescan be found in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang etal. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al.PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard etal. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990)348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982; the entireteachings of which are incorporated herein.

Human monoclonal antibodies of this disclosure can also be preparedusing SCID mice into which human immune cells have been reconstitutedsuch that a human antibody response can be generated upon immunization.Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

The antibodies or antigen-binding portions thereof can be alteredwherein the constant region of the antibody is modified to reduce atleast one constant region-mediated biological effector function relativeto an unmodified antibody. To modify an antibody of the invention suchthat it exhibits reduced binding to the Fc receptor, the immunoglobulinconstant region segment of the antibody can be mutated at particularregions necessary for Fc receptor (FcR) interactions (see, e.g.,Canfield and Morrison (1991) J. Exp. Med. 173:1483-1491; and Lund et al.(1991) J. of Immunol. 147:2657-2662, the entire teachings of which areincorporated herein). Reduction in FcR binding ability of the antibodymay also reduce other effector functions which rely on FcR interactions,such as opsonization and phagocytosis and antigen-dependent cellularcytotoxicity.

Antibody Production

To express an antibody of the invention, DNAs encoding partial orfull-length light and heavy chains are inserted into one or moreexpression vector such that the genes are operatively linked totranscriptional and translational control sequences. (See, e.g., U.S.Pat. No. 6,914,128, the entire teaching of which is incorporated hereinby reference.) In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into a separate vector or, more typically, bothgenes are inserted into the same expression vector. The antibody genesare inserted into an expression vector by standard methods (e.g.,ligation of complementary restriction sites on the antibody genefragment and vector or blunt end ligation if no restriction sites arepresent). Prior to insertion of the antibody or antibody-related lightor heavy chain sequences, the expression vector may already carryantibody constant region sequences. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, a recombinant expression vectorof the invention can carry one or more regulatory sequence that controlsthe expression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, e.g., in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), the entire teaching of which is incorporatedherein by reference. It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., the entireteachings of which are incorporated herein by reference.

In addition to the antibody chain genes and regulatory sequences, arecombinant expression vector of the invention may carry one or moreadditional sequences, such as a sequence that regulates replication ofthe vector in host cells (e.g. origins of replication) and/or aselectable marker gene. The selectable marker gene facilitates selectionof host cells into which the vector has been introduced (see e.g., U.S.Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., theentire teachings of which are incorporated herein by reference). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Suitable selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr-hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

An antibody of the invention can be prepared by recombinant expressionof immunoglobulin light and heavy chain genes in a host cell. To expressan antibody recombinantly, a host cell is transfected with one or morerecombinant expression vectors carrying DNA fragments encoding theimmunoglobulin light and heavy chains of the antibody such that thelight and heavy chains are expressed in the host cell and secreted intothe medium in which the host cells are cultured, from which medium theantibodies can be recovered. Standard recombinant DNA methodologies areused to obtain antibody heavy and light chain genes, incorporate thesegenes into recombinant expression vectors and introduce the vectors intohost cells, such as those described in Sambrook, Fritsch and Maniatis(eds), Molecular Cloning; A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.Nos. 4,816,397 & 6,914,128, the entire teachings of which areincorporated herein.

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is (are) transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like.

Although it is theoretically possible to express the antibodies of theinvention in either prokaryotic or eukaryotic host cells, expression ofantibodies in eukaryotic cells, such as mammalian host cells, issuitable because such eukaryotic cells and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss and Wood (1985)Immunology Today 6:12-13, the entire teaching of which is incorporatedherein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans and Shigella, as well as Bacilli such as B. subtilisand B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published Apr. 12, 1989), Pseudomonas such as P. aeruginosa andStreptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537) and E. coli W3110 (ATCC 27,325) are suitable. These examples areillustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly) and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato and tobacco can also beutilized as hosts.

Suitable mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) PNAS USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inKaufman and Sharp (1982) Mol. Biol. 159:601-621, the entire teachings ofwhich are incorporated herein by reference), NS0 myeloma cells, COScells and SP2 cells. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or secretionof the antibody into the culture medium in which the host cells aregrown. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2), the entire teachings of which are incorporated herein byreference.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants or amplifying the genes encoding the desired sequences.

The host cells used to produce an antibody may be cultured in a varietyof media. Commercially available media such as Ham's F10™ (Sigma),Minimal Essential Medium™ ((MEM), (Sigma), RPMI-1640 (Sigma) andDulbecco's Modified Eagle's Medium™ ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may beused as culture media for the host cells, the entire teachings of whichare incorporated herein by reference. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas gentamycin drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range) andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression and will be apparent to the ordinarilyskilled artisan.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, in certain embodiments it may be desirable totransfect a host cell with DNA encoding either the light chain or theheavy chain (but not both) of an antibody of this invention. RecombinantDNA technology may also be used to remove some or all of the DNAencoding either or both of the light and heavy chains that is notnecessary for binding to the antigen to which the putative antibody ofinterest binds. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than the one to which theputative antibody of interest binds, depending on the specificity of theantibody of the invention, by crosslinking an antibody of the inventionto a second antibody by standard chemical crosslinking methods.

In a suitable system for recombinant expression of an antibody of theinvention, a recombinant expression vector encoding both the antibodyheavy chain and the antibody light chain is introduced into dhfr-CHOcells by calcium phosphate-mediated transfection. Within the recombinantexpression vector, the antibody heavy and light chain genes are eachoperatively linked to CMV enhancer/AdMLP promoter regulatory elements todrive high levels of transcription of the genes. The recombinantexpression vector also carries a DHFR gene, which allows for selectionof CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the antibody heavy andlight chains and intact antibody is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space or directly secreted into themedium. In one aspect, if the antibody is produced intracellularly, as afirst step, the particulate debris, either host cells or lysed cells(e.g., resulting from homogenization), can be removed, e.g., bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems can be firstconcentrated using a commercially available protein concentrationfilter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit.

Prior to the process of the invention, procedures for purification ofantibodies from cell debris initially depend on the site of expressionof the antibody. Some antibodies can be secreted directly from the cellinto the surrounding growth media; others are made intracellularly. Forthe latter antibodies, the first step of a purification processtypically involves: lysis of the cell, which can be done by a variety ofmethods, including mechanical shear, osmotic shock or enzymatictreatments. Such disruption releases the entire contents of the cellinto the homogenate and in addition produces subcellular fragments thatare difficult to remove due to their small size. These are generallyremoved by differential centrifugation or by filtration. Where theantibody is secreted, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit. Where the antibody is secreted into the medium,the recombinant host cells can also be separated from the cell culturemedium, e.g., by tangential flow filtration. Antibodies can be furtherrecovered from the culture medium using the antibody purificationmethods of the invention.

Methods of Treatment Using the Compositions of the Invention

The compositions of the present invention including proteins withcontrolled oligosaccharide heterogeneity, e.g., desired NGA2F and(NGA2F-GlcNAc) and/or NA 1F and NA2F distribution, may be used to treatany disorder in a subject for which the therapeutic protein of interest(e.g., an antibody or an antigen binding portion thereof) comprised inthe composition is appropriate for treating.

A “disorder” is any condition that would benefit from treatment with theprotein of interest. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose thesubject to the disorder in question. In the case of an anti-TNFαantibody or antigen binding portion thereof, such as adalimumab, atherapeutically effective amount of the controlled protein heterogeneitycomposition may be administered to treat a disorder in which TNFαactivity is detrimental.

A disorder in which TNFα activity is detrimental includes a disorder inwhich inhibition of TNFα activity is expected to alleviate the symptomsand/or progression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of TNFα in a biologicalfluid of a subject suffering from the disorder (e.g., an increase in theconcentration of TNFα in serum, plasma, synovial fluid, etc. of thesubject), which can be detected, for example, using an anti-TNFαantibody.

TNFα has been implicated in the pathophysiology of a wide variety of aTNFα-related disorders including sepsis, infections, autoimmunediseases, transplant rejection and graft-versus-host disease (see e.g.,Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024to Moeller et al.; European Patent Publication No. 260 610 B1 byMoeller, A., et al. Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452;Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).Accordingly, the controlled protein heterogeneity composition of theinvention may be used to treat an autoimmune disease, such as rheumatoidarthritis, juvenile idiopathic arthritis or psoriatic arthritis, anintestinal disorder, such as Crohn's disease or ulcerative colitis, aspondyloarthropathy, such as ankylosing spondylitis or a skin disorder,such as psoriasis.

Disorders in which TNFα activity is detrimental are well known in theart and described in detail in U.S. Pat. No. 8,231,876 and U.S. Pat. No.6,090,382, the entire contents of each of which are expresslyincorporated herein by reference. In one embodiment, “a disorder inwhich TNFα activity is detrimental” includes sepsis (including septicshock, endotoxic shock, gram negative sepsis and toxic shock syndrome),autoimmune diseases (including rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis and gouty arthritis, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis, nephrotic syndrome,multisystem autoimmune diseases, lupus (including systemic lupus, lupusnephritis and lupus cerebritis), Crohn's disease and autoimmune hearingloss), infectious diseases (including malaria, meningitis, acquiredimmune deficiency syndrome (AIDS), influenza and cachexia secondary toinfection), allograft rejection and graft versus host disease,malignancy, pulmonary disorders (including adult respiratory distresssyndrome (ARDS), shock lung, chronic pulmonary inflammatory disease,pulmonary sarcoidosis, pulmonary fibrosis, silicosis, idiopathicinterstitial lung disease and chronic obstructive airway disorders(COPD), such as asthma), intestinal disorders (including inflammatorybowel disorders, idiopathic inflammatory bowel disease, Crohn's diseaseand Crohn's disease-related disorders (including fistulas in thebladder, vagina and skin; bowel obstructions; abscesses; nutritionaldeficiencies; complications from corticosteroid use; inflammation of thejoints; erythem nodosum; pyoderma gangrenosum; lesions of the eye,Crohn's related arthralgias, fistulizing Crohn's indeterminant colitisand pouchitis), cardiac disorders (including ischemia of the heart,heart insufficiency, restenosis, congestive heart failure, coronaryartery disease, angina pectoris, myocardial infarction, cardiovasculartissue damage caused by cardiac arrest, cardiovascular tissue damagecaused by cardiac bypass, cardiogenic shock and hypertension,atherosclerosis, cardiomyopathy, coronary artery spasm, coronary arterydisease, valvular disease, arrhythmias and cardiomyopathies),spondyloarthropathies (including ankylosing spondylitis, psoriaticarthritis/spondylitis, enteropathic arthritis, reactive arthritis orReiter's syndrome and undifferentiated spondyloarthropathies), metabolicdisorders (including obesity and diabetes, including type 1 diabetesmellitus, type 2 diabetes mellitus, diabetic neuropathy, peripheralneuropathy, diabetic retinopathy, diabetic ulcerations, retinopathyulcerations and diabetic macrovasculopathy), anemia, pain (includingacute and chronic pains, such as neuropathic pain and post-operativepain, chronic lower back pain, cluster headaches, herpes neuralgia,phantom limb pain, central pain, dental pain, opioid-resistant pain,visceral pain, surgical pain, bone injury pain, pain during labor anddelivery, pain resulting from burns, including sunburn, post partumpain, migraine, angina pain and genitourinary tract-related painincluding cystitis), hepatic disorders (including hepatitis, alcoholichepatitis, viral hepatitis, alcoholic cirrhosis, al antitypsindeficiency, autoimmune cirrhosis, cryptogenic cirrhosis, fulminanthepatitis, hepatitis B and C and steatohepatitis, cystic fibrosis,primary biliary cirrhosis, sclerosing cholangitis and biliaryobstruction), skin and nail disorders (including psoriasis (includingchronic plaque psoriasis, guttate psoriasis, inverse psoriasis, pustularpsoriasis and other psoriasis disorders), pemphigus vulgaris,scleroderma, atopic dermatitis (eczema), sarcoidosis, erythema nodosum,hidradenitis suppurative, lichen planus, Sweet's syndrome, sclerodermaand vitiligo), vasculitides (including Behcet's disease) and otherdisorders, such as juvenile rheumatoid arthritis (JRA), endometriosis,prostatitis, choroidal neovascularization, sciatica, Sjogren's syndrome,uveitis, wet macular degeneration, osteoporosis and osteoarthritis.

As used herein, the term “subject” is intended to include livingorganisms, e.g., prokaryotes and eukaryotes. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats and transgenic non-human animals. In specificembodiments of the invention, the subject is a human.

As used herein, the term “treatment” or “treat” refers to boththerapeutic treatment and prophylactic or preventative measures. Thosein need of treatment include those already with the disorder, as well asthose in which the disorder is to be prevented.

In one embodiment, the invention provides a method of administeringcompositions of an anti-TNFα antibody or antigen binding portionthereof, with desired oligosaccharide heterogeneity, e.g., desired NGA2Fand (NGA2F-GlcNAc) and/or NA1F and NA2F distribution, to a subject suchthat TNFα activity is inhibited or a disorder in which TNFα activity isdetrimental is treated. In one embodiment, the TNFα is human TNFα andthe subject is a human subject. In one embodiment, the anti-TNFαantibody is adalimumab, also referred to as HUMIRA®.

The controlled protein heterogeneity compositions of the invention canbe administered by a variety of methods known in the art. Exemplaryroutes/modes of administration include subcutaneous injection,intravenous injection or infusion. In certain aspects, a controlledprotein heterogeneity composition may be orally administered. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. In certainembodiments, it is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit comprising a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic or prophylactic effect to be achieved and (b)the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a controlled protein heterogeneitycomposition of the invention is 0.01-20 mg/kg or 1-10 mg/kg or 0.3-1mg/kg. With respect to controlled protein heterogeneity compositioncomprising an anti-TNFα antibody or antigen-binding portion thereof,such as adalimumab, an exemplary dose is 40 mg every other week. In someembodiments, in particular for treatment of ulcerative colitis orCrohn's disease, an exemplary dose includes an initial dose (Day 1) of160 mg (e.g., four 40 mg injections in one day or two 40 mg injectionsper day for two consecutive days), a second dose two weeks later of 80mg and a maintenance dose of 40 mg every other week beginning two weekslater. Alternatively, for psoriasis for example, a dosage can include an80 mg initial dose followed by 40 mg every other week starting one weekafter the initial dose.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

Pharmaceutical Formulations Containing the Controlled ProteinHeterogeneity Compositions of the Invention

The present invention further provides preparations and formulationscomprising controlled protein heterogeneity compositions, e.g., havingdesired NGA2F and (NGA2F-GlcNAc) and/or NA1F and NA2F distribution, ofthe invention. It should be understood that any of the proteins ofinterest, such as antibodies and antibody fragments described herein,including proteins of interest having any one or more of the structuraland functional features described in detail throughout the application,may be formulated or prepared as described below. When variousformulations are described in this section as including a protein ofinterest, such as an antibody, it is understood that such a protein ofinterest may be a protein having any one or more of the characteristicsof the proteins of interest described herein. In one embodiment, theantibody is an anti-TNFα antibody or antigen-binding portion thereof.

In certain embodiments, the controlled protein heterogeneitycompositions of the invention may be formulated with a pharmaceuticallyacceptable carrier as pharmaceutical (therapeutic) compositions and maybe administered by a variety of methods known in the art. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. The term“pharmaceutically acceptable carrier” means one or more non-toxicmaterials that do not interfere with the effectiveness of the biologicalactivity of the active ingredients. Such preparations may routinelycontain salts, buffering agents, preservatives, compatible carriers andoptionally other therapeutic agents. Such pharmaceutically acceptablepreparations may also routinely contain compatible solid or liquidfillers, diluents or encapsulating substances which are suitable foradministration into a human. The term “carrier” denotes an organic orinorganic ingredient, natural or synthetic, with which the activeingredient is combined to facilitate the application. The components ofthe pharmaceutical compositions also are capable of being co-mingledwith the proteins of interest (e.g., antibodies) of the presentinvention and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

The controlled protein heterogeneity compositions of the invention arepresent in a form known in the art and acceptable for therapeutic uses.In one embodiment, a formulation of the controlled protein heterogeneitycompositions of the invention is a liquid formulation. In anotherembodiment, a formulation of the controlled protein heterogeneitycompositions of the invention is a lyophilized formulation. In a furtherembodiment, a formulation of the controlled protein heterogeneitycompositions of the invention is a reconstituted liquid formulation. Inone embodiment, a formulation of the controlled protein heterogeneitycompositions of the invention is a stable liquid formulation. In oneembodiment, a liquid formulation of the controlled protein heterogeneitycompositions of the invention is an aqueous formulation. In anotherembodiment, the liquid formulation is non-aqueous. In a specificembodiment, a liquid formulation of the controlled protein heterogeneitycompositions of the invention is an aqueous formulation wherein theaqueous carrier is distilled water.

The formulations of the controlled protein heterogeneity compositions ofthe invention comprise a protein of interest (e.g., an antibody) in aconcentration resulting in a w/v appropriate for a desired dose. Theprotein of interest may be present in the formulation at a concentrationof about 1 mg/ml to about 500 mg/ml, e.g., at a concentration of atleast 1 mg/ml, at least 5 mg/ml, at least 10 mg/ml, at least 15 mg/ml,at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35mg/ml, at least 40 mg/ml, at least 45 mg/ml, at least 50 mg/ml, at least55 mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml, atleast 75 mg/ml, at least 80 mg/ml, at least 85 mg/ml, at least 90 mg/ml,at least 95 mg/ml, at least 100 mg/ml, at least 105 mg/ml, at least 110mg/ml, at least 115 mg/ml, at least 120 mg/ml, at least 125 mg/ml, atleast 130 mg/ml, at least 135 mg/ml, at least 140 mg/ml, at least 150mg/ml, at least 200 mg/ml, at least 250 mg/ml or at least 300 mg/ml.

In a specific embodiment, a formulation of the controlled proteinheterogeneity compositions of the invention comprises at least about 100mg/ml, at least about 125 mg/ml, at least 130 mg/ml or at least about150 mg/ml of protein of interest (e.g., an antibody) of the invention.

In one embodiment, the concentration of protein of interest (e.g.,antibody), which is included in the formulation of the invention, isbetween about 1 mg/ml and about 25 mg/ml, between about 1 mg/ml andabout 200 mg/ml, between about 25 mg/ml and about 200 mg/ml, betweenabout 50 mg/ml and about 200 mg/ml, between about 75 mg/ml and about 200mg/ml, between about 100 mg/ml and about 200 mg/ml, between about 125mg/ml and about 200 mg/ml, between about 150 mg/ml and about 200 mg/ml,between about 25 mg/ml and about 150 mg/ml, between about 50 mg/ml andabout 150 mg/ml, between about 75 mg/ml and about 150 mg/ml, betweenabout 100 mg/ml and about 150 mg/ml, between about 125 mg/ml and about150 mg/ml, between about 25 mg/ml and about 125 mg/ml, between about 50mg/ml and about 125 mg/ml, between about 75 mg/ml and about 125 mg/ml,between about 100 mg/ml and about 125 mg/ml, between about 25 mg/ml andabout 100 mg/ml, between about 50 mg/ml and about 100 mg/ml, betweenabout 75 mg/ml and about 100 mg/ml, between about 25 mg/ml and about 75mg/ml, between about 50 mg/ml and about 75 mg/ml or between about 25mg/ml and about 50 mg/ml.

In a specific embodiment, a formulation of the controlled proteinheterogeneity compositions of the invention comprises between about 90mg/ml and about 110 mg/ml or between about 100 mg/ml and about 210 mg/mlof a protein of interest (e.g., an antibody).

The formulations of the controlled protein heterogeneity compositions ofthe invention comprising a protein of interest (e.g., an antibody) mayfurther comprise one or more active compounds as necessary for theparticular indication being treated, typically those with complementaryactivities that do not adversely affect each other. Such additionalactive compounds are suitably present in combination in amounts that areeffective for the purpose intended.

The formulations of the controlled protein heterogeneity compositions ofthe invention may be prepared for storage by mixing the protein ofinterest (e.g., antibody) having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers,including, but not limited to buffering agents, saccharides, salts,surfactants, solubilizers, polyols, diluents, binders, stabilizers,salts, lipophilic solvents, amino acids, chelators, preservatives or thelike (Goodman and Gilman's The Pharmacological Basis of Therapeutics,12^(th) edition, L. Brunton, et al. and Remington's PharmaceuticalSciences, 16th edition, Osol, A. Ed. (1999)), in the form of lyophilizedformulations or aqueous solutions at a desired final concentration.Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed and includebuffers such as histidine, phosphate, citrate, glycine, acetate andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrolidone; aminoacids such as glycine, glutamine, asparagine, histidine, arginine orlysine; monosaccharides, disaccharides and other carbohydrates includingtrehalose, glucose, mannose or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN, polysorbate 80,PLURONICS™ or polyethylene glycol (PEG).

The buffering agent may be histidine, citrate, phosphate, glycine oracetate. The saccharide excipient may be trehalose, sucrose, mannitol,maltose or raffinose. The surfactant may be polysorbate 20, polysorbate40, polysorbate 80 or Pluronic F68. The salt may be NaCl, KCl, MgCl₂ orCaCl₂

The formulations of the controlled protein heterogeneity compositions ofthe invention may include a buffering or pH adjusting agent to provideimproved pH control. A formulation of the invention may have a pH ofbetween about 3.0 and about 9.0, between about 4.0 and about 8.0,between about 5.0 and about 8.0, between about 5.0 and about 7.0,between about 5.0 and about 6.5, between about 5.5 and about 8.0,between about 5.5 and about 7.0 or between about 5.5 and about 6.5. In afurther embodiment, a formulation of the invention has a pH of about3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2,about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5,about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about8.0, about 8.5 or about 9.0. In a specific embodiment, a formulation ofthe invention has a pH of about 6.0. One of skill in the art understandsthat the pH of a formulation generally should not be equal to theisoelectric point of the particular protein of interest (e.g., antibody)to be used in the formulation.

Typically, the buffering agent is a salt prepared from an organic orinorganic acid or base. Representative buffering agents include, but arenot limited to organic acid salts such as salts of citric acid, ascorbicacid, gluconic acid, carbonic acid, tartaric acid, succinic acid, aceticacid or phthalic acid; Tris, tromethamine hydrochloride or phosphatebuffers. In addition, amino acid components can also function in abuffering capacity. Representative amino acid components which may beutilized in the formulations of the invention as buffering agentsinclude, but are not limited to, glycine and histidine. In certainembodiments, the buffering agent is chosen from histidine, citrate,phosphate, glycine and acetate. In a specific embodiment, the bufferingagent is histidine. In another specific embodiment, the buffering agentis citrate. In yet another specific embodiment, the buffering agent isglycine. The purity of the buffering agent should be at least 98% or atleast 99% or at least 99.5%. As used herein, the term “purity” in thecontext of histidine and glycine refers to chemical purity of histidineor glycine as understood in the art, e.g., as described in The MerckIndex, 13^(th) ed., O'Neil et al. ed. (Merck & Co., 2001).

Buffering agents are typically used at concentrations between about 1 mMand about 200 mM or any range or value therein, depending on the desiredionic strength and the buffering capacity required. The usualconcentrations of conventional buffering agents employed in parenteralformulations can be found in: Pharmaceutical Dosage Form: ParenteralMedications, Volume 1, 2^(nd) Edition, Chapter 5, p. 194, De Luca andBoylan, “Formulation of Small Volume Parenterals”, Table 5: Commonlyused additives in Parenteral Products. In one embodiment, the bufferingagent is at a concentration of about 1 mM or of about 5 mM or of about10 mM or of about 15 mM or of about 20 mM or of about 25 mM or of about30 mM or of about 35 mM or of about 40 mM or of about 45 mM or of about50 mM or of about 60 mM or of about 70 mM or of about 80 mM or of about90 mM or of about 100 mM. In one embodiment, the buffering agent is at aconcentration of 1 mM or of 5 mM or of 10 mM or of 15 mM or of 20 mM orof 25 mM or of 30 mM or of 35 mM or of 40 mM or of 45 mM or of 50 mM orof 60 mM or of 70 mM or of 80 mM or of 90 mM or of 100 mM. In a specificembodiment, the buffering agent is at a concentration of between about 5mM and about 50 mM. In another specific embodiment, the buffering agentis at a concentration of between 5 mM and 20 mM.

In certain embodiments, the formulation of the controlled proteinheterogeneity compositions of the invention comprises histidine as abuffering agent. In one embodiment the histidine is present in theformulation of the invention at a concentration of at least about 1 mM,at least about 5 mM, at least about 10 mM, at least about 20 mM, atleast about 30 mM, at least about 40 mM, at least about 50 mM, at leastabout 75 mM, at least about 100 mM, at least about 150 mM or at leastabout 200 mM histidine. In another embodiment, a formulation of theinvention comprises between about 1 mM and about 200 mM, between about 1mM and about 150 mM, between about 1 mM and about 100 mM, between about1 mM and about 75 mM, between about 10 mM and about 200 mM, betweenabout 10 mM and about 150 mM, between about 10 mM and about 100 mM,between about 10 mM and about 75 mM, between about 10 mM and about 50mM, between about 10 mM and about 40 mM, between about 10 mM and about30 mM, between about 20 mM and about 75 mM, between about 20 mM andabout 50 mM, between about 20 mM and about 40 mM or between about 20 mMand about 30 mM histidine. In a further embodiment, the formulationcomprises about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM,about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mMor about 200 mM histidine. In a specific embodiment, a formulation maycomprise about 10 mM, about 25 mM or no histidine.

The formulations of the controlled protein heterogeneity compositions ofthe invention may comprise a carbohydrate excipient. Carbohydrateexcipients can act, e.g., as viscosity enhancing agents, stabilizers,bulking agents, solubilizing agents and/or the like. Carbohydrateexcipients are generally present at between about 1% to about 99% byweight or volume, e.g., between about 0.1% to about 20%, between about0.1% to about 15%, between about 0.1% to about 5%, between about 1% toabout 20%, between about 5% to about 15%, between about 8% to about 10%,between about 10% and about 15%, between about 15% and about 20%,between 0.1% to 20%, between 5% to 15%, between 8% to 10%, between 10%and 15%, between 15% and 20%, between about 0.1% to about 5%, betweenabout 5% to about 10% or between about 15% to about 20%. In still otherspecific embodiments, the carbohydrate excipient is present at 1% or at1.5% or at 2% or at 2.5% or at 3% or at 4% or at 5% or at 10% or at 15%or at 20%.

Carbohydrate excipients suitable for use in the formulations of theinvention include, but are not limited to, monosaccharides such asfructose, maltose, galactose, glucose, D-mannose, sorbose and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.In one embodiment, the carbohydrate excipients for use in the presentinvention are chosen from, sucrose, trehalose, lactose, mannitol andraffinose. In a specific embodiment, the carbohydrate excipient istrehalose. In another specific embodiment, the carbohydrate excipient ismannitol. In yet another specific embodiment, the carbohydrate excipientis sucrose. In still another specific embodiment, the carbohydrateexcipient is raffinose. The purity of the carbohydrate excipient shouldbe at least 98% or at least 99% or at least 99.5%.

In a specific embodiment, the formulations of the controlled proteinheterogeneity compositions of the invention may comprise trehalose. Inone embodiment, a formulation of the invention comprises at least about1%, at least about 2%, at least about 4%, at least about 8%, at leastabout 20%, at least about 30% or at least about 40% trehalose. Inanother embodiment, a formulation of the invention comprises betweenabout 1% and about 40%, between about 1% and about 30%, between about 1%and about 20%, between about 2% and about 40%, between about 2% andabout 30%, between about 2% and about 20%, between about 4% and about40%, between about 4% and about 30% or between about 4% and about 20%trehalose. In a further embodiment, a formulation of the inventioncomprises about 1%, about 2%, about 4%, about 6%, about 8%, about 15%,about 20%, about 30% or about 40% trehalose. In a specific embodiment, aformulation of the invention comprises about 4%, about 6% or about 15%trehalose.

In certain embodiments, a formulation of the controlled proteinheterogeneity compositions of the invention comprises an excipient. In aspecific embodiment, a formulation of the invention comprises at leastone excipient chosen from: sugar, salt, surfactant, amino acid, polyol,chelating agent, emulsifier and preservative. In one embodiment, aformulation of the invention comprises a salt, e.g., a salt selectedfrom: NaCl, KCl, CaCl₂ and MgCl₂. In a specific embodiment, theformulation comprises NaCl.

A formulation of the controlled protein heterogeneity compositions ofthe invention may comprise at least about 10 mM, at least about 25 mM,at least about 50 mM, at least about 75 mM, at least about 80 mM, atleast about 100 mM, at least about 125 mM, at least about 150 mM, atleast about 175 mM, at least about 200 mM or at least about 300 mMsodium chloride

(NaCl). In a further embodiment, the formulation may comprise betweenabout 10 mM and about 300 mM, between about 10 mM and about 200 mM,between about 10 mM and about 175 mM, between about 10 mM and about 150mM, between about 25 mM and about 300 mM, between about 25 mM and about200 mM, between about 25 mM and about 175 mM, between about 25 mM andabout 150 mM, between about 50 mM and about 300 mM, between about 50 mMand about 200 mM, between about 50 mM and about 175 mM, between about 50mM and about 150 mM, between about 75 mM and about 300 mM, between about75 mM and about 200 mM, between about 75 mM and about 175 mM, betweenabout 75 mM and about 150 mM, between about 100 mM and about 300 mM,between about 100 mM and about 200 mM, between about 100 mM and about175 mM or between about 100 mM and about 150 mM sodium chloride. In afurther embodiment, the formulation may comprise about 10 mM, about 25mM, about 50 mM, about 75 mM, about 80 mM, about 100 mM, about 125 mM,about 150 mM, about 175 mM, about 200 mM or about 300 mM sodiumchloride.

A formulation of the controlled protein heterogeneity compositions ofthe invention may also comprise an amino acid, e.g., lysine, arginine,glycine, histidine or an amino acid salt. The formulation may compriseat least about 1 mM, at least about 10 mM, at least about 25 mM, atleast about 50 mM, at least about 100 mM, at least about 150 mM, atleast about 200 mM, at least about 250 mM, at least about 300 mM, atleast about 350 mM or at least about 400 mM of an amino acid. In anotherembodiment, the formulation may comprise between about 1 mM and about100 mM, between about 10 mM and about 150 mM, between about 25 mM andabout 250 mM, between about 25 mM and about 300 mM, between about 25 mMand about 350 mM, between about 25 mM and about 400 mM, between about 50mM and about 250 mM, between about 50 mM and about 300 mM, between about50 mM and about 350 mM, between about 50 mM and about 400 mM, betweenabout 100 mM and about 250 mM, between about 100 mM and about 300 mM,between about 100 mM and about 400 mM, between about 150 mM and about250 mM, between about 150 mM and about 300 mM or between about 150 mMand about 400 mM of an amino acid. In a further embodiment, aformulation of the invention comprises about 1 mM, 1.6 mM, 25 mM, about50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300mM, about 350 mM or about 400 mM of an amino acid.

The formulations of the controlled protein heterogeneity compositions ofthe invention may further comprise a surfactant. The term “surfactant”as used herein refers to organic substances having amphipathicstructures; namely, they are composed of groups of opposing solubilitytendencies, typically an oil-soluble hydrocarbon chain and awater-soluble ionic group. Surfactants can be classified, depending onthe charge of the surface-active moiety, into anionic, cationic andnonionic surfactants. Surfactants are often used as wetting,emulsifying, solubilizing and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.Pharmaceutically acceptable surfactants like polysorbates (e.g.,polysorbates 20 or 80); polyoxamers (e.g., poloxamer 188); Triton;sodium octyl glycoside; lauryl-, myristyl-, linoleyl- orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, paImidopropyl- or isostearamidopropyl-betaine (e.g.,lauroamidopropyl); myristamidopropyl-, paImidopropyl- orisostearamidopropyl-dimethylamine; sodium methyl cocoyl- or disodiummethyl oleyl-taurate; and the MONAQUA™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol and copolymers ofethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.), canoptionally be added to the formulations of the invention to reduceaggregation. In one embodiment, a formulation of the invention comprisesPolysorbate 20, Polysorbate 40, Polysorbate 60 or Polysorbate 80.Surfactants are particularly useful if a pump or plastic container isused to administer the formulation. The presence of a pharmaceuticallyacceptable surfactant mitigates the propensity for the protein toaggregate. The formulations may comprise a polysorbate which is at aconcentration ranging from between about 0.001% to about 1% or about0.001% to about 0.1% or about 0.01% to about 0.1%. In other specificembodiments, the formulations of the invention comprise a polysorbatewhich is at a concentration of 0.001% or 0.002% or 0.003% or 0.004% or0.005% or 0.006% or 0.007% or 0.008% or 0.009% or 0.01% or 0.015% or0.02%.

The formulations of the controlled protein heterogeneity compositions ofthe invention may optionally further comprise other common excipientsand/or additives including, but not limited to, diluents, binders,stabilizers, lipophilic solvents, preservatives, adjuvants or the like.Pharmaceutically acceptable excipients and/or additives may be used inthe formulations of the invention. Commonly used excipients/additives,such as pharmaceutically acceptable chelators (for example, but notlimited to, EDTA, DTPA or EGTA) can optionally be added to theformulations of the invention to reduce aggregation. These additives areparticularly useful if a pump or plastic container is used to administerthe formulation.

Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (for example, but notlimited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl andthe like), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal or mixtures thereof can optionally beadded to the formulations of the invention at any suitable concentrationsuch as between about 0.001% to about 5% or any range or value therein.The concentration of preservative used in the formulations of theinvention is a concentration sufficient to yield a microbial effect.Such concentrations are dependent on the preservative selected and arereadily determined by the skilled artisan.

Other contemplated excipients/additives, which may be utilized in theformulations of the invention include, for example, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,lipids such as phospholipids or fatty acids, steroids such ascholesterol, protein excipients such as serum albumin (human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,salt-forming counterions such as sodium and the like. These andadditional known pharmaceutical excipients and/or additives suitable foruse in the formulations of the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 21^(st) ed.,Lippincott Williams & Wilkins, (2005) and in the “Physician's DeskReference”, 60^(th) ed., Medical Economics, Montvale, N.J. (2005).Pharmaceutically acceptable carriers can be routinely selected that aresuitable for the mode of administration, solubility and/or stability ofprotein of interest (e.g., an antibody), as well known those in the artor as described herein.

In one embodiment, the controlled protein heterogeneity compositions ofthe invention are formulated with the same or similar excipients andbuffers as are present in the commercial adalimumab (HUMIRA®)formulation, as described in the “Highlights of Prescribing Information”for HUMIRA® (adalimumab) Injection (Revised January 2008) the contentsof which are hereby incorporated herein by reference. For example, eachprefilled syringe of HUMIRA®, which is administered subcutaneously,delivers 0.8 mL (40 mg) of drug product to the subject. Each 0.8 mL ofHUMIRA® contains 40 mg adalimumab, 4.93 mg sodium chloride, 0.69 mgmonobasic sodium phosphate dihydrate, 1.22 mg dibasic sodium phosphatedihydrate, 0.24 mg sodium citrate, 1.04 mg citric acid monohydrate, 9.6mg mannitol, 0.8 mg polysorbate 80 and water for Injection, USP. Sodiumhydroxide is added as necessary to adjust pH.

It will be understood by one skilled in the art that the formulations ofthe controlled protein heterogeneity compositions of the invention maybe isotonic with human blood, wherein the formulations of the inventionhave essentially the same osmotic pressure as human blood. Such isotonicformulations will generally have an osmotic pressure from about 250 mOSmto about 350 mOSm. Isotonicity can be measured by, for example, using avapor pressure or ice-freezing type osmometer. Tonicity of a formulationis adjusted by the use of tonicity modifiers. “Tonicity modifiers” arethose pharmaceutically acceptable inert substances that can be added tothe formulation to provide an isotonicity of the formulation. Tonicitymodifiers suitable for this invention include, but are not limited to,saccharides, salts and amino acids.

In certain embodiments, the formulations of the controlled proteinheterogeneity compositions of the invention have an osmotic pressurefrom about 100 mOSm to about 1200 mOSm or from about 200 mOSm to about1000 mOSm or from about 200 mOSm to about 800 mOSm or from about 200mOSm to about 600 mOSm or from about 250 mOSm to about 500 mOSm or fromabout 250 mOSm to about 400 mOSm or from about 250 mOSm to about 350mOSm.

The concentration of any one component or any combination of variouscomponents, of the formulations of the controlled protein heterogeneitycompositions of the invention is adjusted to achieve the desiredtonicity of the final formulation. For example, the ratio of thecarbohydrate excipient to protein of interest (e.g., antibody) may beadjusted according to methods known in the art (e.g., U.S. Pat. No.6,685,940). In certain embodiments, the molar ratio of the carbohydrateexcipient to protein of interest (e.g., antibody) may be from about 100moles to about 1000 moles of carbohydrate excipient to about 1 mole ofprotein of interest or from about 200 moles to about 6000 moles ofcarbohydrate excipient to about 1 mole of protein of interest or fromabout 100 moles to about 510 moles of carbohydrate excipient to about 1mole of protein of interest or from about 100 moles to about 600 molesof carbohydrate excipient to about 1 mole of protein of interest.

The desired isotonicity of the final formulation may also be achieved byadjusting the salt concentration of the formulations. Pharmaceuticallyacceptable salts and those suitable for this invention as tonicitymodifiers include, but are not limited to, sodium chloride, sodiumsuccinate, sodium sulfate, potassium chloride, magnesium chloride,magnesium sulfate and calcium chloride. In specific embodiments,formulations of the invention comprise NaCl, MgCl₂ and/or CaCl₂. In oneembodiment, concentration of NaCl is between about 75 mM and about 150mM. In another embodiment, concentration of MgCl₂ is between about 1 mMand about 100 mM. Pharmaceutically acceptable amino acids includingthose suitable for this invention as tonicity modifiers include, but arenot limited to, proline, alanine, L-arginine, asparagine, L-asparticacid, glycine, serine, lysine and histidine.

In one embodiment the formulations of the controlled proteinheterogeneity compositions of the invention are pyrogen-freeformulations which are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released only when the microorganisms are brokendown or die. Pyrogenic substances also include fever-inducing,thermostable substances (glycoproteins) from the outer membrane ofbacteria and other microorganisms. Both of these substances can causefever, hypotension and shock if administered to humans. Due to thepotential harmful effects, even low amounts of endotoxins must beremoved from intravenously administered pharmaceutical drug solutions.The Food & Drug Administration (“FDA”) has set an upper limit of 5endotoxin units (EU) per dose per kilogram body weight in a single onehour period for intravenous drug applications (The United StatesPharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). Whentherapeutic proteins are administered in amounts of several hundred orthousand milligrams per kilogram body weight, as can be the case withproteins of interest (e.g., antibodies), even trace amounts of harmfuland dangerous endotoxin must be removed. In certain specificembodiments, the endotoxin and pyrogen levels in the composition areless then 10 EU/mg or less then 5 EU/mg or less then 1 EU/mg or lessthen 0.1 EU/mg or less then 0.01 EU/mg or less then 0.001 EU/mg.

When used for in vivo administration, the formulations of the controlledprotein heterogeneity compositions of the invention should be sterile.The formulations of the invention may be sterilized by varioussterilization methods, including sterile filtration, radiation, etc. Inone embodiment, the protein of interest (e.g., antibody) formulation isfilter-sterilized with a presterilized 0.22-micron filter. Sterilecompositions for injection can be formulated according to conventionalpharmaceutical practice as described in “Remington: The Science &Practice of Pharmacy”, 21^(st) ed., Lippincott Williams & Wilkins,(2005). Formulations comprising proteins of interest (e.g., antibodies),such as those disclosed herein ordinarily will be stored in lyophilizedform or in solution. It is contemplated that sterile compositionscomprising proteins of interest (e.g., antibodies) are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having an adapter that allows retrieval of theformulation, such as a stopper pierceable by a hypodermic injectionneedle. In one embodiment, a composition of the invention is provided asa pre-filled syringe.

In one embodiment, a formulation of the controlled protein heterogeneitycompositions of the invention is a lyophilized formulation. The term“lyophilized” or “freeze-dried” includes a state of a substance that hasbeen subjected to a drying procedure such as lyophilization, where atleast 50% of moisture has been removed.

The phrase “bulking agent” includes a compound that is pharmaceuticallyacceptable and that adds bulk to a lyo cake. Bulking agents known to theart include, for example, carbohydrates, including simple sugars such asdextrose, ribose, fructose and the like, alcohol sugars such asmannitol, inositol and sorbitol, disaccharides including trehalose,sucrose and lactose, naturally occurring polymers such as starch,dextrans, chitosan, hyaluronate, proteins (e.g., gelatin and serumalbumin), glycogen and synthetic monomers and polymers.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest (such as an antibody of the invention), significantly preventsor reduces chemical and/or physical instability of the protein uponlyophilization and subsequent storage. Lyoprotectants include, but arenot limited to, sugars and their corresponding sugar alcohols; an aminoacid such as monosodium glutamate or histidine; a methylamine such asbetaine; a lyotropic salt such as magnesium sulfate; a polyol such astrihydric or higher molecular weight sugar alcohols, e.g., glycerin,dextran, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol;propylene glycol; polyethylene glycol; PLURONICS™; and combinationsthereof. Additional examples of lyoprotectants include, but are notlimited to, glycerin and gelatin and the sugars mellibiose, melezitose,raffinose, mannotriose and stachyose. Examples of reducing sugarsinclude, but are not limited to, glucose, maltose, lactose, maltulose,iso-maltulose and lactulose. Examples of non-reducing sugars include,but are not limited to, non-reducing glycosides of polyhydroxy compoundsselected from sugar alcohols and other straight chain polyalcohols.Examples of sugar alcohols include, but are not limited to,monoglycosides, compounds obtained by reduction of disaccharides such aslactose, maltose, lactulose and maltulose. The glycosidic side group canbe either glucosidic or galactosidic. Additional examples of sugaralcohols include, but are not limited to, glucitol, maltitol, lactitoland iso-maltulose. In specific embodiments, trehalose or sucrose is usedas a lyoprotectant.

The lyoprotectant is added to the pre-lyophilized formulation in a“lyoprotecting amount” which means that, following lyophilization of theprotein in the presence of the lyoprotecting amount of thelyoprotectant, the protein essentially retains its physical and chemicalstability and integrity upon lyophilization and storage.

In one embodiment, the molar ratio of a lyoprotectant (e.g., trehalose)and protein of interest (e.g., antibody) molecules of a formulation ofthe invention is at least about 10, at least about 50, at least about100, at least about 200 or at least about 300. In another embodiment,the molar ratio of a lyoprotectant (e.g., trehalose) and protein ofinterest molecules of a formulation of the invention is about 1, isabout 2, is about 5, is about 10, about 50, about 100, about 200 orabout 300.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized protein of interest (e.g., antibody)formulation in a diluent such that the protein of interest is dispersedin the reconstituted formulation. The reconstituted formulation issuitable for administration (e.g., parenteral administration) to apatient to be treated with the protein of interest and, in certainembodiments of the invention, may be one which is suitable forintravenous administration.

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, such as aformulation reconstituted after lyophilization. In some embodiments,diluents include, but are not limited to, sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution. In an alternative embodiment, diluents can includeaqueous solutions of salts and/or buffers.

In certain embodiments, a formulation of the controlled proteinheterogeneity compositions of the invention is a lyophilized formulationcomprising a protein of interest (e.g., antibody) of the invention,wherein at least about 90%, at least about 95%, at least about 97%, atleast about 98% or at least about 99% of said protein of interest may berecovered from a vial upon shaking said vial for 4 hours at a speed of400 shakes per minute wherein the vial is filled to half of its volumewith the formulation. In another embodiment, a formulation of theinvention is a lyophilized formulation comprising a protein of interestof the invention, wherein at least about 90%, at least about 95%, atleast about 97%, at least about 98% or at least about 99% of the proteinof interest may be recovered from a vial upon subjecting the formulationto three freeze/thaw cycles wherein the vial is filled to half of itsvolume with said formulation. In a further embodiment, a formulation ofthe invention is a lyophilized formulation comprising a protein ofinterest of the invention, wherein at least about 90%, at least about95%, at least about 97%, at least about 98% or at least about 99% of theprotein of interest may be recovered by reconstituting a lyophilizedcake generated from said formulation.

In one embodiment, a reconstituted liquid formulation may comprise aprotein of interest (e.g., antibody) at the same concentration as thepre-lyophilized liquid formulation.

In another embodiment, a reconstituted liquid formulation may comprise aprotein of interest (e.g., antibody) at a higher concentration than thepre-lyophilized liquid formulation, e.g., .about 2 fold, about 3 fold,about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold,about 9 fold or about 10 fold higher concentration of a protein ofinterest than the pre-lyophilized liquid formulation.

In yet another embodiment, a reconstituted liquid formulation maycomprise a protein of interest (e.g., antibody) of the invention at alower concentration than the pre-lyophilized liquid formulation, e.g.,about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold,about 7 fold, about 8 fold, about 9 fold or about 10 fold lowerconcentration of a protein of interest than the pre-lyophilized liquidformulation.

The pharmaceutical formulations of the controlled protein heterogeneitycompositions of the invention are typically stable formulations, e.g.,stable at room temperature.

The terms “stability” and “stable” as used herein in the context of aformulation comprising a protein of interest (e.g., an antibody) of theinvention refer to the resistance of the protein of interest in theformulation to aggregation, degradation or fragmentation under givenmanufacture, preparation, transportation and storage conditions. The“stable” formulations of the invention retain biological activity undergiven manufacture, preparation, transportation and storage conditions.The stability of the protein of interest can be assessed by degrees ofaggregation, degradation or fragmentation, as measured by HPSEC, staticlight scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR),circular dichroism (CD), urea unfolding techniques, intrinsic tryptophanfluorescence, differential scanning calorimetry and/or ANS bindingtechniques, compared to a reference formulation. For example, areference formulation may be a reference standard frozen at −70° C.consisting of 10 mg/ml of a protein of interest of the invention in PBS.

Therapeutic formulations of the controlled protein heterogeneitycompositions of the invention may be formulated for a particular dosage.Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the protein of interest(e.g., antibody) and the particular therapeutic effect to be achievedand (b) the limitations inherent in the art of compounding such aprotein of interest for the treatment of sensitivity in individuals.

Therapeutic compositions of the controlled protein heterogeneitycompositions of the invention can be formulated for particular routes ofadministration, such as oral, nasal, pulmonary, topical (includingbuccal and sublingual), rectal, vaginal and/or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods known in the art ofpharmacy. The amount of active ingredient which can be combined with acarrier material to produce a single dosage form will vary dependingupon the subject being treated and the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the composition which produces a therapeutic effect.By way of example, in certain embodiments, the proteins of interest(including fragments of the protein of interest) are formulated forintravenous administration. In certain other embodiments, the proteinsof interest (e.g., antibodies), including fragments of the proteins ofinterest (e.g., antibody fragments) are formulated for local delivery tothe cardiovascular system, for example, via catheter, stent, wire,intramyocardial delivery, intrapericardial delivery or intraendocardialdelivery.

Formulations of the controlled protein heterogeneity compositions of theinvention which are suitable for topical or transdermal administrationinclude powders, sprays, ointments, pastes, creams, lotions, gels,solutions, patches and inhalants. The active compound may be mixed understerile conditions with a pharmaceutically acceptable carrier and withany preservatives, buffers or propellants which may be required (U.S.Pat. Nos. 7,378,110; 7,258,873; 7,135,180; 7,923,029; and US PublicationNo. 20040042972).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the controlled protein heterogeneity compositions of theinvention may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treatedand like factors well known in the medical arts.

In certain embodiments, the proteins of interest (e.g., antibodies) ofthe invention can be formulated to ensure proper distribution in vivo.For example, the blood-brain barrier (BBB) excludes many highlyhydrophilic compounds. To ensure that the therapeutic compounds of theinvention can cross the BBB (if desired), they can be formulated, forexample, in liposomes. For methods of manufacturing liposomes, see,e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; 5,399,331. The liposomes maycomprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res.Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett.357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);surfactant Protein A receptor (Briscoe et al. (1995) Am. J. Physiol.1233:134), different species of which may comprise the formulations ofthe invention, as well as components of the invented molecules; p 120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; inanother embodiment, the liposomes include a targeting moiety. In anotherembodiment, the therapeutic compounds in the liposomes are delivered bybolus injection to a site proximal to the desired area. Whenadministered in this manner, the composition must be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and may be preserved against thecontaminating action of microorganisms such as bacteria and fungi.Additionally or alternatively, the proteins of interest (e.g.,antibodies) of the invention may be delivered locally to the brain tomitigate the risk that the blood brain barrier slows effective delivery.

In certain embodiments, the controlled protein heterogeneitycompositions of the invention may be administered with medical devicesknown in the art. For example, in certain embodiments a protein ofinterest (e.g., antibody) or a fragment of protein of interest (e.g.,antibody fragment) is administered locally via a catheter, stent, wireor the like. For example, in one embodiment, a therapeutic compositionof the invention can be administered with a needleless hypodermicinjection device, such as the devices disclosed in U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems and modules are known to those skilledin the art.

The efficient dosages and the dosage regimens for the reduced level ofat least one controlled protein heterogeneity composition of theinvention depend on the disease or condition to be treated and can bedetermined by the persons skilled in the art. One of ordinary skill inthe art would be able to determine such amounts based on such factors asthe subject's size, the severity of the subject's symptoms and theparticular composition or route of administration selected.

Alternative Formulations Containing the Controlled Protein HeterogeneityCompositions of the Invention Alternative Aqueous Formulations

The invention also provides a controlled protein heterogeneitycomposition formulated as an aqueous formulation comprising a protein ofinterest and water, as described in U.S. Pat. No. 8,420,081, thecontents of which are hereby incorporated by reference. In these aqueousformulations, the protein of interest is stable without the need foradditional agents. This aqueous formulation has a number of advantagesover conventional formulations in the art, including stability of theprotein of interest in water without the requirement for additionalexcipients, increased concentrations of protein of interest without theneed for additional excipients to maintain solubility of the protein ofinterest and low osmolality. These also have advantageous storageproperties, as the proteins of interest in the formulation remain stableduring storage, e.g., stored as a liquid form for more than 3 months at7° C. or freeze/thaw conditions, even at high protein of interestconcentrations and repeated freeze/thaw processing steps. In oneembodiment, formulations described herein include high concentrations ofproteins of interest such that the aqueous formulation does not showsignificant opalescence, aggregation or precipitation.

In one embodiment, an aqueous controlled protein heterogeneitycomposition comprising a protein of interest, e.g., an antibody, ananti-TNFα antibody or antigen biding portion thereof and water isprovided, wherein the formulation has certain characteristics, such as,but not limited to, low conductivity, e.g., a conductivity of less thanabout 2.5 mS/cm, a protein of interest concentration of at least about10 μg/mL, an osmolality of no more than about 30 mOsmol/kg and/or theprotein of interest has a molecular weight (Mw) greater than about 47kDa. In one embodiment, the formulation has improved stability, such as,but not limited to, stability in a liquid form for an extended time(e.g., at least about 3 months or at least about 12 months) or stabilitythrough at least one freeze/thaw cycle (if not more freeze/thaw cycles).In one embodiment, the formulation is stable for at least about 3 monthsin a form selected from the group consisting of frozen, lyophilized orspray-dried.

In one embodiment, the formulation has a low conductivity, including,for example, a conductivity of less than about 2.5 mS/cm, a conductivityof less than about 2 mS/cm, a conductivity of less than about 1.5 mS/cm,a conductivity of less than about 1 mS/cm or a conductivity of less thanabout 0.5 mS/cm.

In another embodiment, controlled protein heterogeneity compositionsincluded in the formulation have a given concentration, including, forexample, a concentration of at least about 1 mg/mL, at least about 10mg/mL, at least about 50 mg/mL, at least about 100 mg/mL, at least about150 mg/mL, at least about 200 mg/mL or greater than about 200 mg/mL. Inanother embodiment, the formulation of the invention has an osmolalityof no more than about 15 mOsmol/kg.

The aqueous formulations described herein do not rely on standardexcipients, e.g., a tonicity modifier, a stabilizing agent, asurfactant, an anti-oxidant, a cryoprotectant, a bulking agent, alyoprotectant, a basic component and an acidic component. In otherembodiments of the invention, the formulation contains water, one ormore proteins of interest and no ionic excipients (e.g., salts, freeamino acids).

In certain embodiments, the aqueous formulation as described hereincomprise a controlled protein heterogeneity composition comprising aprotein of interest concentration of at least 50 mg/mL and water,wherein the formulation has an osmolality of no more than 30 mOsmol/kg.Lower limits of osmolality of the aqueous formulation are alsoencompassed by the invention. In one embodiment the osmolality of theaqueous formulation is no more than 15 mOsmol/kg. The aqueousformulation of the invention may have an osmolality of less than 30mOsmol/kg and also have a high protein of interest concentration, e.g.,the concentration of the protein of interest is at least 100 mg/mL andmay be as much as 200 mg/mL or greater. Ranges intermediate to the aboverecited concentrations and osmolality units are also intended to be partof this invention. In addition, ranges of values using a combination ofany of the above recited values as upper and/or lower limits areintended to be included.

The concentration of the aqueous formulation as described herein is notlimited by the protein of interest size and the formulation may includeany size range of proteins. Included within the scope of the inventionis an aqueous formulation comprising at least 40 mg/mL and as much as200 mg/mL or more of a protein of interest, for example, 40 mg/mL, 65mg/mL, 130 mg/mL or 195 mg/ml, which may range in size from 5 kDa to 150kDa or more. In one embodiment, the protein of interest in theformulation of the invention is at least about 15 kD in size, at leastabout 20 kD in size; at least about 47 kD in size; at least about 60 kDin size; at least about 80 kD in size; at least about 100 kD in size; atleast about 120 kD in size; at least about 140 kD in size; at leastabout 160 kD in size; or greater than about 160 kD in size. Rangesintermediate to the above recited sizes are also intended to be part ofthis invention. In addition, ranges of values using a combination of anyof the above recited values as upper and/or lower limits are intended tobe included.

The aqueous formulation as described herein may be characterized by thehydrodynamic diameter (D_(h)) of the proteins of interest in solution.The hydrodynamic diameter of the protein of interest in solution may bemeasured using dynamic light scattering (DLS), which is an establishedanalytical method for determining the D_(h) of proteins. Typical valuesfor monoclonal antibodies, e.g., IgG, are about 10 nm. Low-ionicformulations may be characterized in that the D_(h) of the proteins ofinterest are notably lower than protein of interest formulationscomprising ionic excipients. It has been discovered that the D_(h)values of antibodies in aqueous formulations made using thediafiltration/ultrafiltration (DF/UF) process, as described in U.S. Pat.No. 8,420,081, using pure water as an exchange medium, are notably lowerthan the D_(h) of antibodies in conventional formulations independent ofprotein concentration. In one embodiment, antibodies in the aqueousformulation as described herein have a D_(h) of less than 4 nm or lessthan 3 nm.

In one embodiment, the D_(h) of the protein of interest in the aqueousformulation is smaller relative to the D_(h) of the same protein ofinterest in a buffered solution, irrespective of protein of interestconcentration. Thus, in certain embodiments, a protein of interest in anaqueous formulation made in accordance with the methods describedherein, will have a D_(h) which is at least 25% less than the D_(h) ofthe protein of interest in a buffered solution at the same givenconcentration. Examples of buffered solutions include, but are notlimited to phosphate buffered saline (PBS). In certain embodiments,proteins of interest in the aqueous formulation of the invention have aD_(h) that is at least 50% less than the D_(h) of the protein ofinterest in PBS in at the given concentration; at least 60% less thanthe D_(h) of the protein of interest in PBS at the given concentration;at least 70% less than the D_(h) of the protein of interest in PBS atthe given concentration; or more than 70% less than the D_(h) of theprotein of interest in PBS at the given concentration. Rangesintermediate to the above recited percentages are also intended to bepart of this invention, e.g., about 55%, 56%, 57%, 64%, 68% and soforth. In addition, ranges of values using a combination of any of theabove recited values as upper and/or lower limits are intended to beincluded, e.g., about 50% to about 80%.

In one aspect, the aqueous formulation includes the protein of interestat a dosage of about 0.01 mg/kg-10 mg/kg. In another aspect, the dosagesof the protein of interest include approximately 1 mg/kg administeredevery other week or approximately 0.3 mg/kg administered weekly. Askilled practitioner can ascertain the proper dosage and regime foradministering to a subject.

Alternative Solid Unit Formulations

The invention also provides a controlled protein heterogeneitycomposition of the invention formulated as a stable composition of aprotein of interest, e.g., an antibody or antigen binding portionthereof and a stabilizer, referred to herein as solid units, asdescribed in Attorney Docket No. 117813-31001, the contents of which arehereby incorporated by reference herein.

Specifically, it has been discovered that despite having a highproportion of sugar, the solid units comprising the controlled proteinheterogeneity compositions of the invention maintain structural rigidityand resist changes in shape and/or volume when stored under ambientconditions, e.g., room temperature and humidity, for extended periods oftime (e.g., the solid units comprising the controlled proteinheterogeneity compositions of the invention do not require storage in asealed container) and maintain long-term physical and chemical stabilityof the protein of interest without significant degradation and/oraggregate formation. Moreover, despite having a high proportion ofsugar, the solid units comprising the controlled protein heterogeneitycompositions of the invention remain free-flowing when stored underambient conditions, e.g., room temperature and humidity, for extendedperiods of time and yet are easily dissolved in an aqueous solvent,e.g., water (e.g., the solid units require minimal mixing when contactedwith a solvent for reconstitution). Furthermore, the solid unitscomprising the controlled protein heterogeneity compositions of theinvention may be prepared directly in a device for patient use. Theseproperties, when compared to existing techniques which require a vialcontaining a lyophilized protein of interest provided as a cake (whichmay not stabilize a protein of interest for extended periods of time), aseparate vial for a diluent, one or more sterile syringes and severalmanipulation steps, thus provides alternative approaches forreconstitution since the solid units comprising the controlled proteinheterogeneity compositions of the invention may be provided, e.g., in adual chambered cartridge, to make reconstitution invisible duringpatient delivery. Furthermore, the solid units comprising the controlledprotein heterogeneity compositions of the invention are versatile inthat they can be readily and easily adapted for numerous modes ofadministration, such as parenteral and oral administration.

As used herein, the term “solid unit,” refers to a composition which issuitable for pharmaceutical administration and comprises a protein ofinterest, e.g., an antibody or peptide and a stabilizer, e.g., a sugar.The solid unit comprising the controlled protein heterogeneitycompositions of the invention has a structural rigidity and resistanceto changes in shape and/or volume. In one embodiment, the solid unitcomprising the controlled protein heterogeneity compositions of theinvention is obtained by freeze-drying a pharmaceutical formulation of atherapeutic protein of interest. The solid unit comprising thecontrolled protein heterogeneity compositions of the invention may beany shape, e.g., geometric shape, including, but not limited to, asphere, a cube, a pyramid, a hemisphere, a cylinder, a teardrop and soforth, including irregularly shaped units. In one embodiment, the solidunit has a volume ranging from about 1 ml to about 20 ml. In anotherembodiment, the solid unit is not obtained using spray dryingtechniques, e.g., the solid unit is not a powder or granule.

As used herein, the phrase “a plurality of solid units” refers to acollection or population of solid units comprising the controlledprotein heterogeneity compositions of the invention, wherein thecollection comprises two or more solid units having a substantiallyuniform shape, e.g., sphere and/or volume distribution. A substantiallyuniform size distribution is intended to mean that the individual shapesand/or volumes of the solid units comprising the controlled proteinheterogeneity compositions of the invention are substantially similarand not greater than a 10% standard deviation in volume. For example, aplurality of solid units which are spherical in shape would include acollection of solid units having no greater than 10% standard deviationfrom an average volume of the spheres. In one embodiment, the pluralityof solid units is free-flowing.

Kits and Articles of Manufacture Comprising the Controlled ProteinHeterogeneity Compositions of the Invention

Also within the scope of the present invention are kits comprising thecontrolled protein heterogeneity compositions of the invention andinstructions for use. The term “kit” as used herein refers to a packagedproduct comprising components with which to administer the protein ofinterest (e.g., antibody or antigen-binding portion thereof), of theinvention for treatment of a disease or disorder. The kit may comprise abox or container that holds the components of the kit. The box orcontainer is affixed with a label or a Food and Drug Administrationapproved protocol. The box or container holds components of theinvention which may be contained within plastic, polyethylene,polypropylene, ethylene or propylene vessels. The vessels can becapped-tubes or bottles. The kit can also include instructions foradministering a protein of interest (e.g., an antibody) of theinvention.

The kit can further contain one more additional reagents, such as animmunosuppressive reagent, a cytotoxic agent or a radiotoxic agent orone or more additional proteins of interest of the invention (e.g., anantibody having a complementary activity which binds to an epitope inthe TNFα antigen distinct from a first anti-TNFα antibody). Kitstypically include a label indicating the intended use of the contents ofthe kit. The term label includes any writing or recorded materialsupplied on or with the kit or which otherwise accompanies the kit.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with a liquid formulation or lyophilizedformulation of a protein of interest (e.g., an antibody or antibodyfragment thereof) of the invention. In one embodiment, a containerfilled with a liquid formulation of the invention is a pre-filledsyringe. In a specific embodiment, the formulations of the invention areformulated in single dose vials as a sterile liquid. For example, theformulations may be supplied in 3 cc USP Type I borosilicate amber vials(West Pharmaceutical Services—Part No. 6800-0675) with a target volumeof 1.2 mL. Optionally associated with such container(s) can be a noticein the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

In one embodiment, a container filled with a liquid formulation of theinvention is a pre-filled syringe. Any pre-filled syringe known to oneof skill in the art may be used in combination with a liquid formulationof the invention. Pre-filled syringes that may be used are described in,for example, but not limited to, PCT Publications WO05032627,WO08094984, WO9945985, WO03077976, U.S. Pat. No. 6,792,743, U.S. Pat.No. 5,607,400, U.S. Pat. No. 5,893,842, U.S. Pat. No. 7,081,107, U.S.Pat. No. 7,041,087, U.S. Pat. No. 5,989,227, U.S. Pat. No. 6,807,797,U.S. Pat. No. 6,142,976, U.S. Pat. No. 5,899,889, U.S. Pat. No.7,699,811, U.S. Pat. No. 7,540,382, U.S. Pat. No. 7,998,120, U.S. Pat.No. 7,645,267 and US Patent Publication No. US20050075611. Pre-filledsyringes may be made of various materials. In one embodiment apre-filled syringe is a glass syringe. In another embodiment apre-filled syringe is a plastic syringe. One of skill in the artunderstands that the nature and/or quality of the materials used formanufacturing the syringe may influence the stability of a proteinformulation stored in the syringe. For example, it is understood thatsilicon based lubricants deposited on the inside surface of the syringechamber may affect particle formation in the protein formulation. In oneembodiment, a pre-filled syringe comprises a silicone based lubricant.In one embodiment, a pre-filled syringe comprises baked on silicone. Inanother embodiment, a pre-filled syringe is free from silicone basedlubricants. One of skill in the art also understands that small amountsof contaminating elements leaching into the formulation from the syringebarrel, syringe tip cap, plunger or stopper may also influence stabilityof the formulation. For example, it is understood that tungstenintroduced during the manufacturing process may adversely affectformulation stability. In one embodiment, a pre-filled syringe maycomprise tungsten at a level above 500 ppb. In another embodiment, apre-filled syringe is a low tungsten syringe. In another embodiment, apre-filled syringe may comprise tungsten at a level between about 500ppb and about 10 ppb, between about 400 ppb and about 10 ppb, betweenabout 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb,between about 100 ppb and about 10 ppb, between about 50 ppb and about10 ppb, between about 25 ppb and about 10 ppb.

In certain embodiments, kits comprising proteins of interest (e.g.,antibodies) of the invention are also provided that are useful forvarious purposes, e.g., research and diagnostic including forpurification or immunoprecipitation of protein of interest from cells,detection of the protein of interest in vitro or in vivo. For isolationand purification of a protein of interest, the kit may contain anantibody coupled to beads (e.g., sepharose beads). Kits may be providedwhich contain the antibodies for detection and quantitation of a proteinof interest in vitro, e.g., in an ELISA or a Western blot. As with thearticle of manufacture, the kit comprises a container and a label orpackage insert on or associated with the container. The container holdsa composition comprising at least one protein of interest (e.g.,antibody) of the invention. Additional containers may be included thatcontain, e.g., diluents and buffers, control proteins of interest (e.g.,antibodies). The label or package insert may provide a description ofthe composition as well as instructions for the intended in vitro ordiagnostic use.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial, pre-filled syringe or other container that ishermetically sealed. In one embodiment, the unit dosage form is providedas a sterile particulate free solution comprising a protein of interest(e.g., an antibody) that is suitable for parenteral administration. Inanother embodiment, the unit dosage form is provided as a sterilelyophilized powder comprising a protein of interest (e.g., an antibody)that is suitable for reconstitution.

In one embodiment, the unit dosage form is suitable for intravenous,intramuscular, intranasal oral, topical or subcutaneous delivery. Thus,the invention encompasses sterile solutions suitable for each deliveryroute. The invention further encompasses sterile lyophilized powdersthat are suitable for reconstitution.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question, as well as how and how frequently toadminister the pharmaceutical. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, pre-filled syringe, sprayer, insufflator, intravenous (i.v.)bag, envelope and the like; and at least one unit dosage form of apharmaceutical agent contained within said packaging material, whereinsaid pharmaceutical agent comprises a liquid formulation containing aprotein of interest (e.g., an antibody). The packaging material includesinstruction means which indicate how that said protein of interest(e.g., antibody) can be used to prevent, treat and/or manage one or moresymptoms associated with a disease or disorder.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references, including literature references, issued patentsand published patent applications, as cited throughout this applicationare hereby expressly incorporated herein by reference. It should furtherbe understood that the contents of all the figures and tables attachedhereto are expressly incorporated herein by reference. The entirecontents of the following applications are also expressly incorporatedherein by reference:

U.S. Provisional Patent Application 61/893,123, entitled “STABLE SOLIDPROTEIN COMPOSITIONS AND METHODS OF MAKING SAME”, Attorney Docket Number117813-31001, filed on Oct. 18, 2013;

U.S. Provisional Application Ser. No. 61/892,833, entitled “LOW ACIDICSPECIES COMPOSITIONS AND METHODS FOR PRODUCING THE SAME USINGDISPLACEMENT CHROMATOGRAPHY”, Attorney Docket Number 117813-73602, filedon Oct. 18, 2013;

U.S. Provisional Patent Application 61/892,710, entitled “MUTATEDANTI-TNFα ANTIBODIES AND METHODS OF THEIR USE”, Attorney Docket Number117813-73802, filed on Oct. 18, 2013;

U.S. Provisional Patent Application 61/893,068, entitled “LOW ACIDICSPECIES COMPOSITIONS AND METHODS FOR PRODUCING THE SAME”, AttorneyDocket Number 117813-73901, filed on Oct. 18, 2013;

U.S. Provisional Patent Application 61/893,088, entitled “MODULATEDLYSINE VARIANT SPECIES AND METHODS FOR PRODUCING AND USING THE SAME”,Attorney Docket Number 117813-74101, filed on Oct. 18, 2013; and

U.S. Provisional Patent Application 61/893,131, entitled “PURIFICATIONOF PROTEINS USING HYDROPHOBIC INTERACTION CHROMATOGRAPHY”, AttorneyDocket Number 117813-74301, filed on Oct. 18, 2013.

EXAMPLES Example 1 Control of Heterogeneity by Addition of Hydrolysatesto CD Media GIA-1 for Culture of an Adalimumab-Producing CHO Cell Line#1

Control of heterogeneity of therapeutic monoclonal antibodies (mAbs) canaid in ensuring their efficacy, stability, immunogenicity and biologicalactivity. Media composition has been shown to play a role in productquality of mAbs together with process conditions and choice of cellline. In certain embodiments, the present invention provides methods forfine-tuning the product quality profile of a mAb produced in variousChinese hamster ovary (CHO) cell lines by supplementation of yeastand/or plant hydrolysates to chemically defined (CD) media. In certainembodiments, the resulting mAb product is characterized by having adecreased content of complex agalactosylated glycans NGA2F andNGA2F-GlcNac and increased levels of terminally galactosylated glycansNA 1F and NA2F. In certain embodiments, addition of increasing amountsof yeast, soy or wheat hydrolysates from several suppliers to a CDmedium resulted in altered product quality profiles in aconcentration-dependent manner.

In the studies summarized in this example, the effects on glycosylationresulting from the addition of yeast (Bacto TC Yeastolate: 2, 5, 11g/L), soy (BBL Phytone Peptone: 2, 4, 7, 10, 15 g/L) or wheat (WheatPeptone E1: 2, 4, 7, 10, 15 g/L) hydrolysates to CD medium GIA-1 (LifeTechnologies Gibco; proprietary formulation) in the adalimumab-producingCHO cell line #1 were investigated. The antibody produced by theadalimumab-producing CHO cell line #1 was identified as mAb #1.

1.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone or Wheat Peptone E1 according to theexperimental design in FIG. 39. The control cultures were notsupplemented with hydrolysates. In addition to hydrolysates, adaptationmedia was supplemented with 0.876 g/kg L-glutamine and 2.0 mL/kgmethotrexate solution and production media was supplemented with 0.584g/L L-glutamine. The experiment was designed into two blocks. All mediapH was adjusted to approximately 7.1 using 6N hydrochloric acid/5Nsodium hydroxide. The media osmolality was adjusted to 290-300 mOsmol/kgwith sodium chloride.

The adalimumab-producing cultures were expanded for 3 passages (3 dayseach) in their respective adaptation media in a combination of 250 mL(50 mL or 100 mL working volume) and 500 mL (150 mL working volume)Corning vented non-baffled shake flasks and maintained on an orbitalshaker at 110 RPM in a 35° C., 5% CO₂ dry incubator. At each passage,cultures were inoculated at an initial viable cell density (VCD) ofapproximately 0.5×10⁶ cells/mL.

Production cultures were initiated in duplicate 500 mL Corning, vented,non-baffled shake flasks each containing 200 mL culture in dryincubators at 35° C., 5% CO₂ and 110 RPM. Initial VCD was approximately0.5×10⁶ cells/mL. A 1.25% (v/v) 40% glucose stock solution was fed whenthe media glucose concentration was less than 3 g/L.

For all studies described throughout this application, samples werecollected daily and measured for cell density and viability using aCedex cell counter. Retention samples for titer analysis (2×1.5 mL percondition) via Poros A method were collected daily after cultureviability fell below 90%. Samples were centrifuged at 12,000 RPM for 5min and the supernatant was stored at −80° C. until further analysis.The harvest procedure was performed by centrifugation of the culturesample at 3,000 RPM for 30 min followed by storage of the supernatant in125 mL PETG bottles at −80° C. until protein A purification,oligosaccharide and WCX-10 analysis.

For the oligosaccharide assay, the oligosaccharides are released fromthe protein by enzymatic digestion with N-glycanase. Once the glycansare released, the free reducing end of each glycan is labeled byreductive amination with a fluorescent tag, 2-aminobenzamide (2-AB). Theresulting labeled glycans are separated by normal-phase HPLC (NP-HPLC)in acetonitrile: 50 mM ammonium formate, pH 4.4 and detected by afluorescence detector. Quantitation is based on the relative areapercent of detected sugars. The relative area percents of theagalactosyl fucosylated biantennary oligosaccharides (NGA2F and[NGA2F-GlcNac]) and the galactose-containing fucosylated biantennaryoligosaccharides NA1F and NA2F are reported and discussed.

1.2 Culture Growth and Productivity

The majority of cultures grew to a similar peak VCD in the range of9−11×10⁶ cells/mL. Cultures supplemented with 11 g/L yeast hydrolysateBD TC yeastolate experienced slight inhibition of growth (FIG. 1A).Viability profiles were comparable to the control condition withcultures lasting 11 to 13 days (FIG. 1B). Increasing the yeasthydrolysate concentration in CDM media GIA-1 resulted in decreasedaverage productivity compared to the control condition. Culturessupplemented with soy or wheat hydrolysates lasted 12 to 13 days andexperienced slightly increased average titer compared to the controlcondition (FIG. 1C).

1.3 Oligosaccharide Analysis

Addition of yeast, soy or wheat hydrolysates to CD media GIA-1 loweredthe percentage of glycans NGA2F and NGA2F-GlcNac by 1-14% and increasedthe percentage of NA1F and NA2F glycans by 2-12% compared to controlcondition (NGA2F and NGA2F-GlcNac: 89%; NA1F and NA2F: 6%) (FIGS. 2A-B).A dose-dependent decrease in NGA2F and NGA2F-GlcNac and a correspondingincrease in NA1F and NA2F glycans was observed with the addition ofyeast, soy or wheat hydrolysate over the tested range. The highestpercentage decrease in NGA2F and NGA2F-GlcNac and corresponding highestincrease in NA1F and NA2F glycans was recorded for the conditionsupplemented with 7 g/L BD BBL phytone peptone (NGA2F and NGA2F-GlcNac:78% and NA1F and NA2F: 18%) compared to control.

Example 2 Yeast and Soy Hydrolysates Combined Addition to MultipleCommercially Available CD Media for Culture of an Adalimumab-ProducingCHO Cell Line #1

In the study summarized in this example, the effects of combined yeastand soy hydrolysates addition to CD media from multiple suppliers: LifeTechnologies Gibco (OptiCHO and GIA-1), Irvine Scientific (IS CHO-CD)and HyClone/Thermo Scientific (CDM4CHO) on product quality in theadalimumab-producing CHO cell line #1 utilized in Example 1 wereevaluated.

2.1 Materials and Methods

The liquid or powder formulation media were purchased from multiplevendors (Life Technologies Gibco—OptiCHO and GIA-1; Irvine Scientific—ISCHO-CD; and HyClone/Thermo Scientific—CDM4CHO), reconstituted per themanufacturers' recommendations and supplemented with Bacto TC Yeastolateand BBL Phytone Peptone according to the experimental design in FIG. 40.The control cultures for each condition were not supplemented withhydrolysates. All media pH was adjusted to approximately 7.1 using 6Nhydrochloric acid/5N sodium hydroxide.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 110 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended batch-mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

2.2 Culture Growth and Productivity

Commercially available CD media supported markedly different culturegrowth profiles with maximum VCD of 2-9×10⁶ cells/mL and cultureduration ranging from 7 to 15 days (FIG. 3A). Addition of yeast and soyhydrolysates to Life Technologies Gibco OptiCHO and GIA-1 and HyCloneCDM4-CHO media decreased peak VCD and increased culture length by 2 to 6days. However, addition of hydrolysates to Irvine IS CHO-CD mediaincreased peak VCD from 2.5×10⁶ cells/mL to 5.4×10⁶ cells/mL. Cultureviability declined slower with addition of hydrolysates for all mediatested (FIG. 3B). Productivity also varied significantly among cultures;however, the addition of hydrolysates to CD media increased productivityin all cases (FIG. 3C).

2.3 Oligosaccharide Analysis

The combined addition of yeast and soy hydrolysates to variouscommercially available CD media lowered the percentage of NGA2F andNGA2F-GlcNac glycans by 2-10% compared to control (FIG. 4A): from 81% to79% (HyClone CDM4-CHO); from 80% to 75% (Irvine IS CHO-CD); from 88% to80% (Life Technologies OptiCHO); from 90% to 80% (Life TechnologiesGIA-1). The percentage of NA1F and NA2F glycans increased by 3-8%compared to control (FIG. 4B): from 15% to 18% (HyClone CDM4-CHO); from6% to 12% (Life Technologies GIA-1); from 16% to 21% (Irvine IS CHO-CD);from 5% to 13% (Life Technologies OptiCHO).

Example 3 Yeast, Soy or Wheat Hydrolysates Addition to CD Media GIA-1for Culture of an Adalimumab-Producing CHO Cell Line #1

In the study summarized in this example, we investigated the effects onglycosylation resulting from the addition of yeast (5, 11 g/L), soy (4,7 g/L) or wheat (4, 7 g/L) hydrolysates from multiple vendors (BDBiosciences, Sheffield/Kerry Biosciences, DMV International, IrvineScientific and Organotechnie) to CDM GIA-1 in the adalimumab-producingCHO cell line #1.

3.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone or Wheat Peptone E1 according to theexperimental design in FIG. 41. The control cultures were notsupplemented with hydrolysates. All media pH was adjusted toapproximately 7.1 using 6N hydrochloric acid/5N sodium hydroxide. Themedia osmolality was adjusted to 290-300 mOsmol/kg with sodium chloride.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 110 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning, vented, non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

3.2 Culture Growth and Productivity

Culture growth and viability profiles were comparable among all testconditions (FIGS. 5A-C, 6A-C) except for 11 g/L BD Bacto TC yeastolate,for which a slight decrease in the growth rate and maximum VCD wasobserved. Supplementation of CD media GIA-1 with yeast hydrolysateslowered the harvest titer by up to 25% compared to the control, whilethe harvest titer increased up to 14% and 27% with the addition of soyor wheat hydrolysates, respectively (FIG. 7).

3.3 Oligosaccharide Analysis

Addition of yeast, soy or wheat hydrolysates to CD media GIA-1 decreasedthe NGA2F and NGA2F-GlcNac glycans in a dose-dependent manner for allhydrolysate vendors evaluated (FIGS. 8A-B). Addition of yeasthydrolysates to CD media GIA-1 lowered the percentage of NGA2F andNGA2F-GlcNac glycans by 4-9% and increased the percentage of NA1F andNA2F glycans by 5-10% compared to control (NGA2F and NGA2F-GlcNac: 90%;NA1F and NA2F: 6%). Addition of soy hydrolysates to CD media GIA-1decreased the NGA2F and NGA2F-GlcNac glycans by 9-14% and increased theNA1F and NA2F glycans by 11-15% compared to control. Addition of wheathydrolysates decreased the NGA2F and NGA2F-GlcNac glycans by 4-11% andincreased the NA1F and NA2F glycans by 6-12% compared to control.

Example 4 Control of Heterogeneity by Addition of Reduced Ratio of Yeastto Plant Hydrolysate

To identify the role which the ratio of yeast to plant hydrolysate playsin connection with the generation of protein heterogeneity, experimentsemploying a range of different hydrolysate ratios were undertaken. Thecell culture medium employed in each experimental process contains bothyeast and soy hydrolysate (phytone). The ratios of yeast to soyhydrolysate (by weight) are 1.55, 0.67 and 0.25. The total weight ofyeastolate and soy hydrolysate were not changed in each experimentalprocess. Two distinct yeastolate lots were used in connection with theseexperiments (see FIGS. 9 & 11 and 10 & 12, respectively). Culturegrowth, productivity and product quality were assessed. As outlined inFIGS. 9-12, reducing the yeast to soy hydrolysate ratio resulted inaltered oligosaccharide profiles.

4.1. Materials and Methods

The CHO cell line #1 was employed in the studies covered here. Theproduction medium used in this experiment contains basal medium PFCHO,Bacto TC yeastolate and phytone peptone. The pH of all media wasadjusted to 7.15; and media osmolality was adjusted to 373-403 mOsmol/kgwith sodium chloride. For each experiment, 500 mL shakers with 200 mLworking volume were employed at the following conditions: 35° C.constant temperature; 5% CO₂; and 110 RPM. Cultures were inoculated atan initial viable cell density (VCD) of approximately 0.5×10⁶ cells/mL.Two mL of 40% w/w glucose solution was added to each shaker when theglucose concentration dropped below 2 g/L. The shakers were harvestedwhen the viable cell density decreased to approximately 50%. The harvestbroth was centrifuged at 3200 rpm for 30 min at 5° C. to remove cellsand the supernatant was stored at −80° C.

Samples were taken daily from each shaker to monitor growth. Thefollowing equipment was used to analyze the samples: Cedex cell counter,Radiometer blood gas analyzer, YSI glucose analyzer and osmometer. Theharvest samples stored at −80° C. were later thawed and analyzed fortiter with Poros A HPLC method. In addition, the thawed samples werefiltered through a 0.2 μm filter, purified by Protein A chromatographyand then oligosaccharide analysis was performed as described in Example1.

4.2 Cell Growth and Productivity

In the first hydrolysate study, the viable cell densities for thereduced ratios of yeastolate to phytone (i.e. Y/P=0.67 and Y/P=0.25)were much lower than the viable cell density for the 1.55 ratio ofyeastolate to phytone (FIG. 9). As a result, the IVCC on day 13 (i.e.the harvest day) was significantly lower for the reduced ratioconditions compared to the 1.55 ratio condition and the titer was alsolower (but not statistically significantly—data not shown). Theviability profiles were comparable until day 8 (FIG. 9). After day 8,the viability declined faster for the reduced ratio conditions. Inhydrolysate study 2, the viable cell density and viability for the 1.55ratio were slightly lower than those with reduced ratio in theexponential phase, but higher in the decline phase (FIG. 10). However,the titer for the 1.55 ratio shaker was 0.2 g/L lower than the reducedratio (i.e. Y/P=0.67) (data not shown).

4.3. Oligosaccharide Analysis

Glycosylation profiles for hydrolysate studies 1 and 2 are shown inFIGS. 11 and 12, respectively. Reducing the ratio of yeastolate tophytone reduced the percentage of NGA2F and (NGA2F-GlcNAc) glycan. Inhydrolysate study 1, the percentage of NGA2F and (NGA2F-GlcNAc) wassignificantly reduced for Y/P=0.67 and Y/P=0.25 as compared to Y/P=1.55.The p values were 0.03 and 0.001 for Y/P=0.67 and Y/P=0.25,respectively. At the same time, the percentage of NA 1F and NA2F wasincreased significantly as the ratio of yeastolate to phytone wasreduced.

As shown in FIG. 12 in hydrolysate study 2, the difference in thepercentage of NGA2F and (NGA2F-GlcNAc) between Y/P=0.67 and Y/P=1.55 wassignificant (i.e. p=0.000002). The percentage of NGA2F and(NGA2F-GlcNAc) was lowered from 77.5% in the 1.55 ratio to approximately75.4% with the reduced ratio.

Therefore, this study successfully demonstrated that reducing the ratioof yeastolate to phytone could alter oligosaccharide profile using twodifferent lots of yeast hydrolysate.

Example 5 Control of Heterogeneity by Supplementation with Asparagine

The present invention relates to methods for modulating theglycosylation profile of a monoclonal antibody (mAb) by varying theconcentration of asparagine in cell culture media. Cell culture mediumcomponents, such as asparagine, are commonly used and are typicalconstituents of suspension culture media. These nutrients are importantfor ensuring both robust cell growth and production of glycoproteins. Ithas been shown that the cell viability and product titer can be enhancedby the addition of asparagine to a glutamine-free production medium(Genentech, Inc. “Production of Proteins in Glutamine-Free Cell CultureMedia” WO2011019619 (2010)). However, the present invention providesmethods to modify glycosylation distribution by adjusting theconcentration of asparagine. Without being bound by theory, it isthought that the effect of asparagine on glycosylation profile of anantibody is through its conversion to glutamine and/or aspartate.Asparagine is the amide donor for glutamine and can be converted toglutamine and/or aspartate (H Huang, Y Yu, X Yi, Y Zhang “Nitrogenmetabolism of asparagine and glutamate in Vero cells studied by 1H/15 NNMR spectroscopy” Applied microbiology and biotechnology 77 (2007)427-436). Glutamine and aspartate are important intermediates inpyrimidine synthesis; and it is known that enhancing de novo pyrimidinebiosynthesis could increase intracellular UTP concentration (Genentech,Inc. “Galacosylation of Recombinant Glycoproteins” US20030211573(2003)). In addition, studies have suggested that glutamine andaspartate limitation is expected to inhibit amino sugar synthesis (G BNyberg, R R Balcarcel, B D Follstad, G Stephanopoulos, D I Wang“Metabolic effects on recombinant interferon-gamma glycosylation incontinuous culture of Chinese hamster ovary cells” Biotechnology andBioengineering 62 (1999) 336-47; D C F Wong, K T K Wong, L T Goh, C KHeng, M G S. Yap “Impact of dynamic online fed-batch strategies onmetabolism, productivity and N-glycosylation quality in CHO cellcultures” Biotechnology and Bioengineering 89 (2005) 164-177). Both UTPand amino sugar are required for the synthesis of UDP-GlcNac, which isthe substrate for protein glycosylation process. It is also possiblethat the effect of asparagine on glycosylation is via increasing ammoniaconcentration in the cell culture medium since it is showed that theaddition of ammonia in CHO cultures could reduce the extent ofglycosylation of synthesized EPO (M. Yang and M. Butler “Effect ofAmmonia on the Glycosylation of Human Recombinant Erythropoietin inCulture” Biotechnol. Prog. 16 (2000) 751-759). We have found thatammonia concentration was increased after asparagine addition into thecell culture media.

In the studies summarized in Example 5, we investigated the effects onproduct quality attributes resulting from the addition of asparagine tohydrolysate based medium in an adalimumab-producing CHO cell line,generically named CHO cell line #1. Two experiments were performed inthe instant Example. For the first experiment, glutamine and/orasparagine were added (at an individual concentration of 0.4 g/L) on day6. For the second experiment, asparagine was added at different dosage(i.e. 0.4 g/L, 0.8 g/L or 1.6 g/L) either on day 0 (before inoculation)or together with the first glucose shot (happened on day 7).

5.1 Materials and Methods

The CHO cell line #1 was employed in the studies covered here. Uponthaw, cells were expanded in a 19-days seed train and then transferredinto seed reactors for up to 7 days in growth medium. The cells werethen brought to the laboratory and used in the small scale bioreactorexperiments. The media used in these experiments contains basal mediaPFCHO (proprietary formulation), Bacto TC Yeastolate and PhytonePeptone.

Three litter Applikon bioreactors were sterilized and then charged withproduction medium. At inoculation, cells were aseptically transferredinto each bioreactor to reach an initial cell density of 0.5×10⁶ viablecells/mL. After inoculation, the bioreactors were set to the followingconditions: pH=7.1, T=35° C., DO=30% and agitation=200 rpm. The pH wasshifted from 7.1 to 6.9 over the first 2.5 days and held at 6.9 for theremainder of the run. The percentage of dissolved oxygen was controlledby sparging a mixture of air and oxygen. The addition of 0.5 N NaOH orsparging of CO₂ maintained the pH. When the glucose concentration fellbelow 2 g/L, approximately 1.25% (v/v) of glucose solution (400 g/kg)was added to the cell culture.

For the first experiment, glutamine and/or asparagine were added (at anindividual concentration of 0.4 g/L) together with the first glucoseshot (happened on day 6). For the second experiment, asparagine wasadded at different dosage (i.e., 0.4 g/L, 0.8 g/L or 1.6 g/L) either onday 0 (before inoculation) or together with the first glucose shot(happened on day 7).

Samples were taken daily from each reactor to monitor growth. Thefollowing equipment was used to analyze the samples: Cedex cell counterfor cell density and viability; Radiometer ABL 5 blood gas analyzer forpH, pCO2 and pO2; YSI 7100 analyzer for glucose and lactateconcentration. Some of the daily samples and the harvest samples werecentrifuged at 3,000 RPM for 30 min and then the supernatants werestored at −80° C. Later, the thawed harvest samples were filteredthrough a 0.2 μm filter, purified by Protein A chromatography and thenoligosaccharide analysis was performed and then oligosaccharide analysiswas performed as described in Example 1.

5.2 Culture Growth and Productivity

In both of the experiments performed in 3 L bioreactor in hydrolysatebased media with CHO cell line #1 described in the instant Example, theaddition of glutamine and/or asparagine together with a glucose shotincreased the maximum cell density (FIGS. 13A and 15A, respectively).The increase in cell density is started two days after the addition inboth cases. Maximum viable cell density was consistent when 0.4 g/L ofglutamine or asparagine was added. Increasing the concentration ofasparagine to 0.8 g/L or adding both glutamine and asparagine at aconcentration of 0.4 g/L each further increased the maximum viable celldensity; however, adding asparagine at a higher concentration than 0.8g/L (e.g., 1.6 g/L) did not continue to increase the maximum viable celldensity. In contrast, when asparagine was added on day 0 (beforeinoculation), the maximum viable cell density increased in a dosedependent manner, with the maximum viable cell density being reachedwhen 1.6 g/L of asparagine was added on day 0 (FIG. 17A).

A drop in viability was delayed, as compared to control cultures, inboth experiments described in the instant Example for approximately 3days when glutamine and/or asparagine was added on day 6 or 7 (FIGS. 13Band 15B, respectively). However, the drop in viability accelerated onthe last day of the cultures. In contrast, although the drop inviability was delayed when asparagine was added on day 0, the effect ofdelaying viability decay was not as efficient as when the amino acidswere added later (e.g., on day 6 or day 7) as shown in FIG. 17B.

5.3 Oligosaccharide Analysis

The experiments described in the instant Example indicate thatoligosaccharide distribution is altered with the addition of asparagineand/or glutamine. The addition of asparagine increased NGA2F andNGA2F-GlcNac in a dose dependent manner. Compared to control, thepercentage of NGA2F and NGA2F-GlcNac was increased by 1.0-3.9% and thepercentage of NA1F and NA2F was decreased by 1.1-4.3% when 0.4 to 1.6g/L asparagine was added on either day 0 or days 6 or 7 (FIGS. 14A-14B,16A-16B and 18A-18B). Addition of 0.4 g/L glutamine increased thepercentage of NGA2F and NGA2F-GlcNac by 0.7% and lowered the percentageof NA1F and NA2F by 0.9%. Adding both asparagine and glutamine (0.4 g/Lof each) increased the percentage of NGA2F and NGA2F-GlcNAc by 3.3% anddecreased the percentage of NA1F and NA2F by 4.2%. In addition, the cellgrowth profile is the same when 0.8 and 1.6 g/L of asparagine was addedon day? (FIGS. 15A and 15B), but a dose dependent effect onoligosaccharide distribution was observed (FIGS. 16A and 16B),indicating that the effect on oligosaccharide distribution was due tothe addition of asparagine and not the increased maximum viable celldensity or delayed drop in viability.

Example 6 Yeast, Soy or Wheat Hydrolysate Addition to CommerciallyAvailable CD Media is CHO-CD for Culture of an Adalimumab-Producing CHOCell Line #1

In the study summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia IS CHO-CD (Irvine Scientific) in the adalimumab-producing CHO cellline #1 utilized in Example 1 were evaluated.

6.1 Materials and Methods

Adaptation and production media (Irvine Scientific IS CHO-CD 91119) weresupplemented with Bacto TC Yeastolate, BBL Phytone Peptone or WheatPeptone E1 according to the experimental design in FIG. 42. The controlcultures were not supplemented with hydrolysates. All media pH wasadjusted to approximately 7.1.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 110 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

6.2 Culture Growth and Productivity

Addition of yeast, soy or wheat hydrolysates to Irvine IS CHO-CD mediaincreased the maximum VCD and culture length for most conditions studiedcompared to the control (FIG. 19A). The largest increase in maximum VCDwas recorded for cultures supplemented with 5 g/L Bacto TC Yeastolate. Aconcentration-dependent increase in harvest titer was observed for allcultures supplemented with hydrolysates (FIG. 19C).

6.3 Oligosaccharide Analysis

Supplementation of Irvine IS CHO-CD media with yeast hydrolysatesdecreased the percentage of NGA2F and NGA2F-GlcNac glycans by 3-4% andincreased the percentage of NA1F and NA2F glycans by the same percentagecompared to control (NGA2F and NGA2F-GlcNac: 73%; NA1F and NA2F: 25%)(FIGS. 20A-B). Addition of soy hydrolysates to Irvine IS CHO-CD mediadecreased the percentage of NGA2F and NGA2F-GlcNac glycans by 4% andincreased the percentage of NA1F and NA2F glycans by the same percentagecompared to control. However, addition of wheat hydrolysates to IrvineIS CHO-CD media resulted in an opposite trend. A concentration-dependentincrease in the percentage of NGA2F and NGA2F-GlcNac glycans by 1-3% anda corresponding decrease in the percentage of NA1F and NA2F glycans wasobserved.

Example 7 Yeast, Soy or Wheat Hydrolysate Addition to CD Media GIA-1 forCulture of an Adalimumab-Producing CHO Cell Line #2

In the study summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia GIA-1 in an adalimumab-producing CHO cell line, generically namedCHO cell line #2 were evaluated. The antibody produced by theadalimumab-producing CHO cell line #2 was identified as mAb #2.

7.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone or Wheat Peptone E1 according to theexperimental design in FIG. 43. The control cultures were notsupplemented with hydrolysates. All media pH was adjusted toapproximately 7.1 and the media osmolality was adjusted to 290-300mOsmol/kg.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 180 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

7.2 Culture Growth and Productivity

Supplementation of yeast, soy or wheat hydrolysates to CD media GIA-1extended the culture length by 1 to 3 days and decreased the maximum VCDin a dose-dependent manner (FIGS. 21A-B). The addition of thesehydrolysates at the highest concentrations significantly decreasedmaximum VCD, with wheat hydrolysates added at 10 g/L showing the mostsevere growth inhibition effects. However, an impact on harvest titerwas only observed for the culture supplemented with 10 g/L wheathydrolysates (65% reduction). An increase in the harvest titer comparedto the control (FIG. 21C) was found in most other cultures.

7.3 Oligosaccharide Analysis

Addition of yeast hydrolysates decreased the percentage of NGA2F andNGA2F-GlcNac glycans by 3-5% and increased the percentage of NA1F andNA2F glycans by 5-8% compared to control (NGA2F and NGA2F-GlcNac: 89%;NA1F and NA2F: 3%) (FIGS. 22A-B). Addition of soy hydrolysates to CDmedia GIA-1 decreased the NGA2F and NGA2F-GlcNac glycans by 8-12% andincreased the NA1F and NA2F glycans by 10-15% compared to control.Addition of wheat hydrolysates decreased the NGA2F and NGA2F-GlcNacglycans by 6-7% and increased the NA1F and NA2F glycans by 9-10%compared to control.

Example 8 Yeast, Soy or Wheat Hydrolysate Addition to CD Media GIA-1 forCulture of an Adalimumab-Producing CHO Cell Line #3

In the study summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia GIA-1 in an adalimumab-producing CHO cell line, generically namedCHO cell line #3 were evaluated.

8.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone or Wheat Peptone E1 according to theexperimental design in FIG. 44. The control cultures were notsupplemented with hydrolysates. All media pH was adjusted toapproximately 7.1 and the media osmolality was adjusted to 290-300mOsmol/kg.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 140 RPM in a 36° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

8.2 Culture Growth and Productivity

Supplementation of production CD media with high concentrations ofhydrolysates—11 g/L yeast, 15 g/L soy or 15 g/L wheat hydrolysates,decreased the culture growth rate and increased the culture lengthcompared to the control (FIGS. 23A-B). Harvest titer increased withincreasing hydrolysate concentrations in the production media, exceptfor the condition supplemented with 15 g/L wheat hydrolysates, whichexperienced significant growth inhibition and harvest titer decreasecompared to control (FIG. 23C).

8.3 Oligosaccharide Analysis

Supplementation of CD media GIA-1 with yeast, soy or wheat hydrolysatesdecreased the percentage of NGA2F and NGA2F-GlcNac glycans and increasedthe percentage of NA 1F and NA2F glycans in a dose-dependent manner(FIGS. 24A-B). Addition of yeast hydrolysates decreased the percentageof NGA2F and NGA2F-GlcNac glycans by 5-12% and increased the percentageof NA1F and NA2F glycans by 3-11% compared to control (NGA2F andNGA2F-GlcNac: 91%; NA1F and NA2F: 6%). Addition of soy hydrolysates toCD media GIA-1 decreased the NGA2F and NGA2F-GlcNac glycans by 13-25%and increased the NA1F and NA2F glycans by 13-25% compared to control.Addition of wheat hydrolysates decreased the NGA2F and NGA2F-GlcNacglycans by 12-18% and increased the NA1F and NA2F glycans by 12-18%compared to control.

Example 9 Yeast, Soy or Wheat Hydrolysate Addition to CD Media GIA-1 forCulture of a CHO Cell Line Producing mAb #1

In the studies summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia GIA-1 in a CHO cell line producing mAb #1 were evaluated.

9.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate (BD Biosciences; catalog #255772), BBL Phytone Peptone (BDBiosciences; catalog #211096) or Wheat Peptone E1 (Organotechnie;catalog #19559) according to the experimental design in FIG. 45. Thecontrol cultures were not supplemented with hydrolysates. All media pHwas adjusted to approximately 7.2 and the media osmolality was adjustedto 290-330 mOsmol/kg.

Cultures were expanded for 4 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an Infors Multitron orbital shaker at 140RPM in a 36° C., 5% CO₂ incubator. Production cultures were initiated induplicate 500 mL (200 mL working volume) Corning vented non-baffledshake flasks at approximately 1.0×10⁶ cells/mL initial VCD. The studywas run in an extended-batch mode by feeding a glucose solution (1.0%(v/v) of 40% solution) when the media glucose concentration fell below 3g/L.

9.2 Culture Growth and Productivity

Supplementation of yeast, soy or wheat hydrolysates to the CD mediaGIA-1 did not affect culture growth profiles dramatically (FIGS. 25A-B).There was some dose-dependent reduction of the peak VCD compared tocontrol as the hydrolysate concentrations increased, particularly in thecase of soy hydrolysates, but overall the growth profiles were similar.However, the culture duration was extended to 11-14 days compared to 9days for control. Cultures supplemented with 11 g/L yeast hydrolysatehad a substantial increase in harvest titer (FIG. 25C) that far exceededthe other conditions.

9.3 Oligosaccharide Analysis

Addition of yeast hydrolysates to CD media GIA-1 lowered the percentageof NGA2F and NGA2F-GlcNac glycans by 3% and increased the percentage ofNA1F and NA2F glycans by 4% compared to control (NGA2F and NGA2F-GlcNac:92%; NA1F and NA2F: 5%)

(FIGS. 26A-B). Addition of soy hydrolysates lowered the percentage ofNGA2F and NGA2F-GlcNac glycans by 7-13% and increased the percentage ofNA1F and NA2F glycans by 8-12% compared to control. Addition of wheathydrolysates lowered the percentage of NGA2F and NGA2F-GlcNac glycans by5-8% and increased the percentage of NA1F and NA2F glycans by 6-9%compared to control.

Example 10 Yeast, Soy or Wheat Hydrolysate Addition to CD Media GIA-1for Culture of a CHO Cell Line Producing mAb #2

In the study summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia GIA-1 in a CHO cell line producing mAb #2 were evaluated.

10.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TC, BBLPhytone Peptone or Wheat Peptone E1 according to the experimental designin FIG. 46. The control cultures were not supplemented withhydrolysates. All media pH was adjusted to approximately 7.2 and themedia osmolality was adjusted to 280-330 mOsmol/kg.

Upon thaw, cells were cultured in CD media GIA-1 growth media in acombination of Corning vented non-baffled shake flasks and maintained ona shaker platform at 140 RPM and L cell bags. Cultures were propagatedin a 35° C., 5% CO₂ dry incubator. Production cultures were initiated induplicate 500 mL (200 mL working volume) Corning vented non-baffledshake flasks at an initial VCD of approximately 0.5×10⁶ cells/mL. Theshake flask study was run in an extended-batch mode by feeding a glucosesolution (1.25% (v/v) of 40% solution) when the media glucoseconcentration fell below 3 g/L. For this study, samples were collecteddaily and measured for cell density and viability using a NOVA cellcounter.

10.2 Culture Growth and Productivity

Supplementation of yeast, soy or wheat hydrolysates to CD media GIA-1did not impact culture growth for most conditions studied compared tocontrol (FIG. 27A). Supplementation with hydrolysates led to higherviability profiles compared to control (FIG. 27B). The addition of wheathydrolysates increased harvest titer compared to the control (FIG. 27C).

10.3 Oligosaccharide Analysis

Addition of yeast hydrolysates to CD media GIA-1 lowered the percentageof NGA2F and NGA2F-GlcNac glycans by 3% (FIG. 28A) and increased thepercentage of NA1F and NA2F glycans by 7% (FIG. 28B) in a dose-dependentmanner compared to control (NGA2F and NGA2F-GlcNac: 75%; NA1F and NA2F:8%). Addition of soy hydrolysates lowered the percentage of NGA2F andNGA2F-GlcNac by 2-12% and increased the percentage of NA1F and NA2F by4-16% compared to control (NGA2F and NGA2F-GlcNac: 76%; NA1F and NA2F:11%). For this cell line, there was no significant difference in thepercentage of NGA2F and NGA2F-GlcNac glycans between the controlcondition and the cultures supplemented with wheat hydrolysates at theconcentration range evaluated. Furthermore, only a minor increase in thepercentage of NA1F and NA2F glycans was observed.

Example 11 Combined Yeast, Soy and/or Wheat Hydrolysate Addition to CDMedia GIA-1 for Culture of an Adalimumab-Producing CHO Cell Line #1

In the study summarized in this example, the effects on glycosylationresulting from the individual or combined addition of yeast, soy and/orwheat hydrolysates to CD media GIA-1 in the adalimumab-producing CHOcell line #1 utilized in Example 1 were evaluated.

11.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone and/or Wheat Peptone E1 according to theexperimental design in FIGS. 47 and 48. The control cultures were notsupplemented with hydrolysates. All media pH was adjusted toapproximately 7.1 and the media osmolality was adjusted to 290-300mOsmol/kg.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 110 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

11.2 Culture Growth and Productivity

Supplementation of yeast, soy and/or wheat hydrolysates to CD mediaGIA-1 resulted in slight growth inhibition and reduced maximum VCDcompared to the control (FIG. 29A). Culture viability profiles andharvest titer were comparable for all cultures (FIGS. 29B-C).

11.3 Oligosaccharide Analysis

Supplementation of yeast hydrolysates only to CD media GIA-1 decreasedthe percentage of NGA2F and NGA2F-GlcNac glycans by 4% and increased thepercentage of NA1F and NA2F glycans by 6% compared to control (NGA2F andNGA2F-GlcNac: 90%; NA1F and NA2F: 4%) (FIGS. 30A-B). Supplementation ofsoy hydrolysates only decreased the percentage of NGA2F and NGA2F-GlcNacglycans by 7% and increased the percentage of NA1F and NA2F glycans by9% compared to control. Supplementation of wheat hydrolysates decreasedthe percentage of NGA2F and NGA2F-GlcNac glycans only by 5% andincreased the percentage of NA1F and NA2F glycans by 8% compared tocontrol.

The addition of two hydrolysates (yeast and soy; yeast and wheat; soyand wheat) further decreased the percentage of NGA2F and NGA2F-GlcNacglycans and increased the percentage of NA1F and NA2F glycans by acouple of percentages compared to the addition of each componentindividually (FIGS. 30A-B). Supplementing CD media GIA-1 with all threehydrolysates did not result in any further changes in the glycosylationprofile, indicating a saturation state being reached.

Example 12 Effect of Asparagine in CD Media GIA-1 for Culture ofAdalimumab-Producing CHO Cell Line #1

In the study summarized in this Example, the effects on product qualityattributes resulting from the addition of asparagine to CD media GIA-1in an adalimumab-producing CHO cell line, generically named CHO cellline #1 were investigated.

12.1 Materials and Methods

The CHO cell line #1 was employed in the study covered here. Upon thaw,cells were expanded in a 19-days seed train and then transferred intoseed reactors for up to 7 days in growth medium. The cells were thenbrought to the laboratory and adapted in 500-mL shaker flasks with 200mL working volume in CD media GIA1 medium for 13 days with 3 passages.The shaker flasks were placed on a shaker platform at 110 RPM in a 35°C., 5% CO₂ incubator.

The chemically defined growth or production media, was prepared frombasal IVGN CD media GIA1. For preparation of the IVGN CD mediaformulation, the proprietary media was supplemented with L-glutamine,insulin, sodium bicarbonate, sodium chloride and methotrexate solution.Production media consisted of all the components in the growth medium,excluding methotrexate. In addition, 5 mM of Galactose (Sigma, G5388)and 101.1M of Manganese (Sigma, M1787) were supplemented into productionmedium. Osmolality was adjusted by the concentration of sodium chloride.All media was filtered through filter systems (0.22 μm PES) and storedat 4° C. until usage.

Production cultures were initiated in duplicate 500 mL Corning, vented,non-baffled shaker flasks each containing 200 mL culture in dryincubators with 5% CO₂ at 35° C. and 110 RPM. Initial VCD wasapproximately 0.5×10⁶ cells/ml. The shake flask study was run in anextended batch mode by feeding a glucose solution (1.25% (v/v) of 40%solution) when the media glucose concentration fell below 3 g/L.Asparagine stock solution (20 g/L) was fed to culture on Day 6 toincrease Asparagine concentration by 0, 0.4, 1.2 and 2.0 g/L.

Samples were taken daily from each reactor to monitor growth. Thefollowing equipment was used to analyze the samples: Cedex cell counterfor cell density and viability; YSI 7100 analyzer for glucose andlactate concentration.

Some of the daily samples and the harvest samples were centrifuged at3,000 rpm for 30 min and then supernatants were stored at −80° C. Thethawed harvest samples were subsequently filtered through a 0.2 μmfilter, purified by Protein A chromatography and then oligosaccharideanalysis was performed as described in Example 1.

12.2 Culture Growth and Productivity

Feeding of asparagine to CD media GIA-1 did not impact culture growthfor most conditions studied as compared to the control (FIG. 31A). Thecultures showed similar growth rates and reached maximum VCD of ˜12×10⁶cells/mL. Culture viabilities were also very similar to that of thecontrols (FIG. 31B). Similarly, all the cultures examined here resultedin comparable harvest titers of approximately 1.7 g/L (FIG. 31C).

12.3 Oligosaccharide Analysis

The effect of asparagine addition on oligosaccharide distribution wasconsistent with the experiments performed in hydrolysate based mediadescribed above. The addition of asparagine increased NGA2F andNGA2F-GlcNac glycans in a dose dependent manner (FIG. 32A). Thepercentage of NGA2F and NGA2F-GlcNac in the control sample (withoutAsparagine addition) was as low as 74.7%. In the sample with theaddition of asparagine the percentage of NGA2F and NGA2F-GlcNAc wasincreased to 76.1% (0.4 g/L of asparagine), 79.2% (1.2 g/L ofasparagine) and 79.0% (2.0 g/L of asparagine), for a total increase of4.5%.

The percentage of NA1F and NA2F in the control sample (withoutasparagine addition) was as high as 22.3% (FIG. 32B). In the sample withthe addition of asparagine the percentage of NA1F and NA2F was decreasedto 21.1% (0.4 g/L of asparagine), 17.8% (1.2 g/L of asparagine) and17.8% (2.0 g/L of asparagine), for a total reduction of 4.5%.

Example 13 Effect of Asparagine in CD Media GIA-1 for Culture ofAdalimumab-Producing CHO Cell Line #3

In the study summarized in Example 13, the effects on product qualityattributes resulting from the addition of asparagine to CD media GIA-1in an adalimumab-producing CHO cell line, generically named CHO cellline #3 were investigated.

13.1 Materials and Methods

The CHO cell line #3 was employed in the study covered here. Upon thaw,adalimumab producing cell line #3 was cultured in CD media GIA-1 in acombination of vented shake flasks on a shaker platform @ 140 rpm and 20L wave bags. Cultures were propagated in a 36° C., 5% CO₂ incubator toobtain the required number of cells to be able to initiate productionstage cultures.

The chemically defined growth or production media was prepared frombasal IVGN CD media GIA1. For preparation of the IVGN CD mediaformulation, the proprietary media was supplemented with L-glutamine,sodium bicarbonate, sodium chloride and methotrexate solution.Production media consisted of all the components in the growth medium,excluding methotrexate. In addition, 10 mM of Galactose (Sigma, G5388)and 0.2 μM of Manganese (Sigma, M1787) were supplemented into productionmedium. Osmolality was adjusted by the concentration of sodium chloride.All media was filtered through filter systems (0.22 μm PES) and storedat 4° C. until usage.

Production cultures were initiated in duplicate 500 mL Corning, vented,non-baffled shaker flasks each containing 200 mL culture in dryincubators with 5% CO₂ at 36° C. and 140 RPM. Initial VCD wasapproximately 0.5×10⁶ cells/ml. The shake flask study was run in anextended batch mode by feeding a glucose solution (1.25% (v/v) of 40%solution) when the media glucose concentration fell below 3 g/L.Asparagine stock solution (20 g/L) was fed to culture on Day 6 toincrease asparagine concentration by 0, 0.4, 0.8, 1.2, 1.6 and 2.0 g/L.

Samples were taken daily from each reactor to monitor growth. Thefollowing equipment was used to analyze the samples: Cedex cell counterfor cell density and viability; YSI 7100 analyzer for glucose andlactate concentration.

Some of the daily samples and the harvest samples were centrifuged at3,000 rpm for 30 min and then supernatants were stored at −80° C. Thethawed harvest samples were subsequently filtered through a 0.2 μmfilter, purified by Protein A chromatography and then oligosaccharideanalysis was performed as described in Example 1.

13.2 Culture Growth and Productivity

The experiment described in the instant Example used a different cellline (i.e., CHO cell line #3) in CD media GIA-1. Culture growth andviability profiles were comparable among all test conditions withdifferent dosage of asparagine added on day 6 (FIGS. 33A and 33B). Allcultures reached maximum VCD of ˜18−19×10⁶ cells/mL. The product titer(˜1.5-1.6 g/L) was slightly reduced when higher dosage of asparagine wasadded (FIG. 33C).

13.3 Oligosaccharide Analysis

Again, the addition of asparagine increased NGA2F and NGA2F-GlcNac (FIG.34A). The percentage of NGA2F and NGA2F-GlcNac in the control sample(without asparagine addition) was as low as 68.7%. In the sample withthe addition of asparagine, the percentage of NGA2F and NGA2F-GlcNac wasincreased by 4.1-5.1% when 0.4 to 2.0 g/L asparagine was added on day 6(FIG. 34A). The percentage of NA1F and NA2F in the control sample(without asparagine addition) was as high as 25.6% (FIG. 34B). In thesample with the addition of asparagine the percentage of NA1F and NA2Fwas decreased by 3.8-4.6% when 0.4 to 2.0 g/L asparagine was added onday 6 (FIG. 34B).

Example 14 Effect of Asparagine in a Shaker Flask Batch Culture in CDMedia GIA-1 with a CHO Cell Line Producing mAb #2

In the studies summarized in Example 14, the effects on product qualityattributes resulting from the addition of asparagine to CD media GIA-1from Life Technologies Gibco in a CHO cell line producing monoclonalantibody #2 were investigated. In this instant Example, asparagine waseither supplemented into culture media during media preparation or addedon day 5 of the cell culture process.

14.1 Materials and Methods

mAb #2 producing cell line was employed in the study covered here. Uponthaw, cells were cultured in chemically defined growth media in acombination of vented baffled shake flasks (Corning) on a shakerplatform at 140 RPM. All media pH was adjusted to approximately 7.2 andthe media osmolality was adjusted to 280-330 mOsmol/kg.

Cultures were propagated in a 35° C., 5% CO₂ incubator to obtain therequired number of cells to be able to initiate production stagecultures. Production cultures were initiated in duplicate 500 mL ventednon-baffled Corning shake flasks (200 mL working volume) at an initialviable cell density (VCD) of approximately 0.5×10⁶ cells/mL. The shakeflask study was run in an extended batch mode by feeding a glucosesolution (1.25% (v/v) of 40% solution) when the media glucoseconcentration fell below 3 g/L. Asparagine (Sigma, Catalog Number A4284)were solubilized in Milli-Q water to make a 30 g/L stock solution. Allmedia was filtered through Corning or Millipore 1 L filter systems (0.22μm PES) and stored at 4° C. until usage.

For asparagine supplemented into culture media during media preparation,asparagine stock solution was supplemented to production media toincrease asparagine concentration by 0, 0.4, 0.8 and 1.6 g/L. Afteraddition of asparagine, media was brought to a pH similar tonon-supplemented (control) media using 5N hydrochloric acid/5N NaOH andit was brought to an osmolality similar to non-supplemented (control)media by adjusting the concentration of sodium chloride. For asparagineaddition study, asparagine stock solution was added to culture on Day 5to increase Asparagine concentration by 0, 0.4, 0.8 and 1.6 g/L.

For all studies described throughout this invention, samples werecollected daily and measured for cell density and viability using a NOVAcell counter. Retention samples for titer analysis via Poros A methodwere collected by centrifugation at 12,000 RPM for 5 min when theculture viability began declining. The cultures were harvested bycollecting 125 mL aliquots and centrifuging at 3,000 RPM for 30 min whenculture viability was near or below 50%. All supernatants were stored at−80° C. until analysis. The harvest samples were Protein A purified andthen oligosaccharide analysis was performed as described in Example 1.

14.2 Culture Growth and Productivity

Adding asparagine to CD media GIA-1 during medium preparation or on day5 of the cell culture did not impact culture growth for most conditionsstudied as compared to the non-supplemented 0 g/L controls (FIGS. 45Aand 47A). The cultures showed similar growth rates and reached maximumVCD of 22−24×10⁶ cells/mL. Culture viabilities were also very similar tothat of the controls (FIGS. 35B and 37B). Similarly, all the culturesexamined here resulted in comparable harvest titers of approximately 0.9g/L of mAb #2 (FIGS. 35C and 37C).

14.3 Oligosaccharide Analysis

The addition of asparagine during medium preparation increased NGA2F andNGA2F-GlcNac glycans in a dose dependent manner (FIG. 36A). Thepercentage of NGA2F and NGA2F-GlcNac in the control sample (withoutasparagine addition) was as low as 76.3%. In the sample with theaddition of asparagine the percentage of NGA2F and NGA2F-GlcNac wasincreased to 81.5% (0.4 g/L of asparagine), 85.5% (0.8 g/L ofasparagine) and 85.9% (1.6 g/L of asparagine), for a total increase of9.6%. The percentage of NA1F and NA2F in the control sample (withoutasparagine addition) was as high as 11.5% (FIG. 36B). In the sample withthe addition of asparagine the percentage of NA1F and NA2F was decreasedto 9.8% (0.4 g/L of asparagine), 7.8% (0.8 g/L of asparagine) and 7.0%(1.6 g/L of asparagine), for a total reduction of 4.5%. With mAb #2 cellline used in the study, the percentage of Mannose type glycans was alsodecreased with the supplementation of asparagine. The percentage ofMannoses in the control sample (without asparagine addition) was as highas 12.2% (FIG. 36B). In the sample with the addition of asparagine thepercentage of Mannoses was decreased to 8.6% (0.4 g/L of asparagine),6.7% (0.8 g/L of asparagine) and 7.1% (1.6 g/L of asparagine), for atotal reduction of 5.5%.

The addition of asparagine on day 5 of the culture also increased NGA2Fand NGA2F-GlcNac glycans in a dose dependent manner (FIG. 38A). Thepercentage of NGA2F and NGA2F-GlcNac in the control sample (withoutasparagine addition) was as low as 79.7%. In the sample with theaddition of asparagine the percentage of NGA2F and NGA2F-GlcNac wasincreased to 80.5% (0.4 g/L of asparagine), 82.1% (0.8 g/L ofasparagine) and 84.1% (1.6 g/L of asparagine), for a total increase of4.4%. The percentage of NA1F and NA2F in the control sample (withoutasparagine addition) was as high as 9.7% (FIG. 38B). In the sample withthe addition of asparagine the percentage of NA 1F and NA2F wasdecreased to 9.4% (0.4 g/L of asparagine), 9.6% (0.8 g/L of asparagine)and 8.5% (1.6 g/L of asparagine), for a total reduction of 1.2%. Again,the percentage of Mannose type glycans was also decreased with thesupplementation of asparagine. The percentage of Mannoses in the controlsample (without asparagine addition) was as high as 10.6% (FIG. 38B). Inthe sample with the addition of asparagine the percentage of Mannoseswas decreased to 10.1% (0.4 g/L of asparagine), 8.3% (0.8 g/L ofasparagine) and 7.4% (1.6 g/L of asparagine), for a total reduction of3.2%.

Example 15 Effect of PEA Hydrolysate Addition to CD Media GIA-1 inAdalimumab-Producing CHO Cell Line #1

In the study summarized in this Example, the effects on glycosylationresulting from the addition of pea hydrolysate (Hy-Pea 7404, Kerry: 2,4, 7, 10 g/L) to chemically defined (CD) medium GIA-1 (Life TechnologiesGibco) in the adalimumab-producing CHO cell line #1 was investigated.

15.1 Materials and Methods

Adaptation media was supplemented with pea hydrolysate at aconcentration of 2 g/L and production media were supplemented with peahydrolysate at concentrations of 2, 4, 7, 10 g/L. Cultures notsupplemented with pea hydrolysate were included as a control. Inaddition to pea hydrolysate, adaptation media was supplemented with0.876 g/kg L-glutamine and 2.0 mL/kg methotrexate solution; productionmedia was supplemented with 0.584 g/L L-glutamine. The pH of productionmedia was adjusted to approximately 7.1 using 6N hydrochloric acid/5Nsodium hydroxide. The media osmolality was adjusted to approximately 315mOsmol/kg with sodium chloride.

The adalimumab-producing cultures were expanded for 4 passages (3 dayseach) in adaptation media containing 2 g/L pea hydrolysate in acombination of 250 mL (50 mL or 100 mL working volume), 500 mL (150 mLworking volume) and 1 L (300 mL working volume) Corning ventednon-baffled shake flasks and maintained on an orbital shaker at 110 RPMin a 35° C., 5% CO₂ dry incubator. At each passage, cultures wereinoculated at an initial viable cell density (VCD) of approximately0.5×10⁶ cells/mL.

Production cultures were initiated in duplicate 500 mL Corning, vented,non-baffled shake flasks each containing 200 mL culture in dryincubators at 35° C., 5% CO₂ and 110 RPM. Initial VCD was approximately0.5×10⁶ cells/mL. A 1.25% (v/v) 40% glucose stock solution was fed whenthe media glucose concentration was less than 3 g/L.

Samples were collected and measured for cell density and viability asset forth in Example 1. In addition, the oligosaccharide assay wasperformed as set forth in Example 1.

15.2 Culture Growth and Productivity

The majority of cultures grew to a similar peak VCD in the range of9−10×10⁶ cells/mL. Cultures supplemented with 10 g/L pea hydrolysateexperienced slight inhibition of growth (FIG. 49A). The viabilityprofile of cultures with 2 g/L pea hydrolysate were comparable to thecontrol condition; however, a small dose-dependent decrease in viabilitywas observed with higher concentrations of hydrolysate. The cultureduration (12 days) was similar within the hydrolysate concentrationrange evaluated (FIG. 49B). However, a dose-dependent decrease in cellproductivity compared to the control condition was observed with theaddition of pea hydrolysate (FIG. 49C).

15.3 Oligosaccharide Analysis

Addition of pea hydrolysate to CD media GIA-1 lowered the percentage ofNGA2F and NGA2F-GlcNac glycans by 11-15% and increased the percentage ofNA1F and NA2F glycans by 12-16% compared to control condition (NGA2F andNGA2F-GlcNac: 91%; NA1F and NA2F: 4%) (FIGS. 50A-B). A dose-dependentdecrease in NGA2F and NGA2F-GlcNac and a corresponding increase in NA1Fand NA2F glycans was observed with the addition of pea hydrolysate overthe tested range.

1. A method for controlling the oligosaccharide distribution of arecombinantly-expressed protein sample comprising supplementing a cellculture medium used in the recombinant expression of said protein with ayeast hydrolysate and/or a plant hydrolysate.
 2. The method of claim 1,wherein the recombinantly-expressed protein is an antibody or anantigen-binding portion thereof.
 3. The method of claim 2, wherein theantibody is an anti-TNFα antibody.
 4. The method of claim 3, wherein theanti-TNFα antibody is adalimumab.
 5. The method of claim 1, wherein theyeast hydrolysate is selected from the group consisting of Bacto TCYeastolate, HyPep Yeast Extract and UF Yeast Hydrolysate.
 6. The methodof claim 1, wherein the plant hydrolysate is selected from the groupconsisting of a soy hydrolysate, a wheat hydrolysate, a ricehydrolysate, a cotton seed hydrolysate, a pea hydrolysate, a cornhydrolysate and, a potato hydrolysate.
 7. The method of claim 1, whereinthe plant hydrolysate is selected from the group consisting of BBLPhytone Peptone, HyPep 1510, SE50 MAF-UF, UF Soy Hydrolysate, WheatPeptone E1, HyPep 4601 and Proyield WGE80M Wheat.
 8. The method of claim1, wherein the cell culture medium is supplemented with yeasthydrolysate to achieve a yeast hydrolysate concentration from about 2g/L to about 11 g/L.
 9. The method of claim 1, wherein the cell culturemedium is supplemented with yeast hydrolysate to achieve a yeasthydrolysate concentration of about 2 g/L, 5 g/L or 11 g/L.
 10. Themethod of claim 1, wherein the cell culture medium is supplemented withplant hydrolysate to achieve a plant hydrolysate concentration fromabout 2 g/L to about 15 g/L.
 11. The method of claim 1, wherein the cellculture medium is supplemented with plant hydrolysate to achieve a planthydrolysate concentration of about 2 g/L, 4 g/L, 7 g/L, 10 g/L or 15g/L.
 12. The method of claim 1, wherein the cell culture medium issupplemented with yeast hydrolysate and plant hydrolysate to achieve ayeast hydrolysate to plant hydrolysate ratio of about 0.1 to about 4.0.13. The method of claim 1, wherein the cell culture medium issupplemented with yeast hydrolysate and plant hydrolysate to achieve ayeast hydrolysate to plant hydrolysate ratio of about 0.25 to about1.55.
 14. The method of claim 1, wherein the recombinantly-expressedprotein sample is produced by a CHO cell line.
 15. The method of claim1, wherein supplementing the cell culture medium with yeast hydrolysateand/or plant hydrolysate decreases the percentage of oligosaccharidesNGA2F and (NGA2F-GlcNAc) present in the protein sample.
 16. The methodof claim 15, wherein supplementing the cell culture medium with yeasthydrolysate and/or plant hydrolysate decreases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) by at least about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%.
 17. Themethod of claim 15, wherein supplementing the cell culture medium withyeast hydrolysate and/or plant hydrolysate decreases the percentage ofoligosaccharides NGA2F and (NGA2F-GlcNAc) by about 1%-30%, 2%-25%,5%-20% or 5%-15%.
 18. The method of claim 15, wherein supplementing thecell culture medium with yeast hydrolysate and/or plant hydrolysatedecreases the percentage of oligosaccharides NGA2F and (NGA2F-GlcNAc) inthe protein sample to about 64%-88%, 70%-88% or 75%-85%.
 19. The methodof claim 1, wherein supplementing the cell culture medium with yeasthydrolysate and/or plant hydrolysate increases the percentage ofoligosaccharides NA1F and NA2F present in the protein sample.
 20. Themethod of claim 19, wherein supplementing the cell culture medium withyeast hydrolysate and/or plant hydrolysate increases the percentage ofoligosaccharides NA1F and NA2F by at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%.
 21. The method of claim19, wherein supplementing the cell culture medium with yeast hydrolysateand/or plant hydrolysate increases the percentage of oligosaccharidesNA1F and NA2F by about 1%-30%, 2%-25%, 5%-20% or 5%-15%.
 22. The methodof claim 19, wherein supplementing the cell culture medium with yeasthydrolysate and/or plant hydrolysate increases the percentage ofoligosaccharides NA1F and NA2F in the protein sample to about 8%-31%,10%-25% or 10%-20%.
 23. The method of claim 1, wherein the cell culturemedium comprises yeast and/or plant hydrolysate prior to supplementingthe medium with the yeast hydrolysate and/or plant hydrolysate.
 24. Themethod of claim 1, wherein the cell culture medium is substantially freeof yeast and/or plant hydrolysate prior to supplementing the medium withthe yeast hydrolysate and/or plant hydrolysate.
 25. A method forcontrolling the oligosaccharide distribution of arecombinantly-expressed protein sample comprising modulating theasparagine and/or glutamine concentration of the cell culture mediumused in the recombinant expression of said protein.
 26. The method ofclaim 25, wherein the recombinantly-expressed protein is an antibody oran antigen-binding portion thereof.
 27. The method of claim 26, whereinthe antibody is an anti-TNFα antibody.
 28. The method of claim 27,wherein the anti-TNFα antibody is adalimumab.
 29. The method of claim25, wherein the recombinantly-expressed protein is produced in a CHOcell line.
 30. The method of claim 25, comprising modulating theconcentration of glutamine and asparagine.
 31. The method of claim 25,comprising increasing the concentration of asparagine.
 32. The method ofclaim 25, comprising increasing the concentration of glutamine.
 33. Themethod of claim 25, wherein the concentration of asparagine and/orglutamine in the cell culture medium is modulated to a level of greaterthan about 0.2 g/L.
 34. The method of claim 25, wherein theconcentration of asparagine and/or glutamine in the cell culture mediumis modulated to a level of greater than about 0.4 g/L, 0.6 g/L, 0.8 g/L,1.0 g/L, 1.2 g/L, 1.4 g/L, 1.6 g/L, 1.8 g/L or 2 g/L.
 35. The method ofclaim 25, wherein the concentration of asparagine and/or glutamine inthe cell culture medium is modulated to a level between about 0.4g/L-1.4 g/L.
 36. The method of claim 25, wherein the cell culture mediumcomprises hydrolysate.
 37. The method of claim 36, wherein thehydrolysate is a yeast hydrolysate and/or a plant hydrolysate.
 38. Themethod of claim 37, wherein the yeast hydrolysate is selected from thegroup consisting of Bacto TC Yeastolate, HyPep Yeast Extract and UFYeast Hydrolysate.
 39. The method of claim 37, wherein the planthydrolysate is selected from the group consisting of a soy hydrolysate,a wheat hydrolysate, a rice hydrolysate, a cotton seed hydrolysate, apea hydrolysate, a corn hydrolysate and a potato hydrolysate.
 40. Themethod of claim 37, wherein the plant hydrolysate is selected from thegroup consisting of BBL Phytone Peptone, HyPep 1510, SE50 MAF-UF, UF SoyHydrolysate, Wheat Peptone E1, HyPep 4601 and Proyield WGE80M Wheat. 41.The method of claim 25, wherein an increase in the concentration ofasparagine and/or glutamine in the cell culture medium increases thepercentage of oligosaccharides NGA2F and (NGA2F-GlcNAc) present in theprotein sample.
 42. The method of claim 41, wherein an increase in theconcentration of asparagine and/or glutamine in the cell culture mediumincreases the percentage of oligosaccharides NGA2F and (NGA2F-GlcNAc) byat least about 0.1%, 0.2%, 0.3%. 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%,1.5%, 2%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or20%.
 43. The method of claim 25, wherein an increase in theconcentration of asparagine and/or glutamine in the cell culture mediumincreases the percentage of oligosaccharides NGA2F and (NGA2F-GlcNAc) byabout 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%,6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 11%, 12%, 13%,14% or 15%.
 44. The method of claim 25, wherein an increase in theconcentration of asparagine and/or glutamine in the cell culture mediumincreases the percentage of oligosaccharides NGA2F and (NGA2F-GlcNAc) byabout 0.5%-15%, by about 0.5%-10% or by about 4-6%.
 45. The method ofclaim 25, wherein an increase in the concentration of asparagine and/orglutamine in the cell culture medium decreases the percentage ofoligosaccharides NA1F and NA2F present in the protein sample.
 46. Themethod of claim 25, wherein an increase in the concentration ofasparagine and/or glutamine in the cell culture medium decreases thepercentage of oligosaccharides NA1F and NA2F by at least about 0.5%,1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%,7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 11%, 12%, 13%, 14% or 15%.47. The method of claim 25, wherein an increase in the concentration ofasparagine and/or glutamine in the cell culture medium decreases thepercentage of oligosaccharides NA1F and NA2F by about 0.5%-10%, by about0.5%-6% or by about 2-5%.
 48. The method of claim 25, wherein the cellculture medium comprises asparagine and/or glutamine prior to modulatingthe concentration of asparagine and/or glutamine.
 49. The method ofclaim 25, wherein the cell culture medium is substantially free ofasparagine and/or glutamine prior to modulating the concentration ofasparagine and/or glutamine.
 50. A composition comprising therecombinantly expressed protein produced by the methods of any one ofclaim 1 or
 25. 51. The composition of claim 50, wherein the protein isan anti-TNFα antibody or an antigen-binding portion thereof.
 52. Thecomposition of claim 51, wherein the anti-TNFα antibody is adalimumab.53. A pharmaceutical composition comprising the composition of claim 50,and a pharmaceutically acceptable excipient.
 54. A compositioncomprising N-linked glycosylated adalimumab, wherein theoligosaccharides NGA2F and (NGA2F-GlcNAc) are present at about 64%-88%and/or wherein the oligosaccharides NA1F and NA2F are present at about8-31%, based on the total amount of oligosaccharides present in thecomposition.
 55. The composition of claim 54, wherein theoligosaccharides NGA2F and (NGA2F-GlcNAc) are present at about 70%-88%.56. The composition of claim 54, wherein the oligosaccharides NGA2F and(NGA2F-GlcNAc) are present at about 75%-85%.
 57. The composition ofclaim 54, wherein the oligosaccharides NA1F and NA2F are present atabout 10%-25%.
 58. The composition of claim 54, wherein theoligosaccharides NA1F and NA2F are present at about 10%-20%.
 59. Apharmaceutical composition comprising the composition of claim 54, and apharmaceutically acceptable excipient.
 60. A composition comprisingN-linked glycosylated adalimubab, wherein oligosaccharides NA1F and NA2Fare present at greater than about 27% based on the total amount ofoligosaccharides present in the composition.
 61. The composition ofclaim 60, wherein oligosaccharides NA1F and NA2F are present at greaterthan about 29% based on the total amount of oligosaccharides present inthe composition.
 62. The composition of claim 60, whereinoligosaccharides NA1F and NA2F are present at about 27%-31% based on thetotal amount of oligosaccharides present in the composition.
 63. Thecomposition of claim 60, wherein oligosaccharides NA1F and NA2F arepresent at about 29%-31% based on the total amount of oligosaccharidespresent in the composition.
 64. A composition comprising N-linkedglycosylated adalimumab, wherein oligosaccharides NA1F and NA2F arepresent at less than about 20% based on the total amount ofoligosaccharides present in the composition.
 65. The composition ofclaim 64, wherein oligosaccharides NA1F and NA2F are present at about10% to about 20% based on the total amount of oligosaccharides presentin the composition.
 66. A pharmaceutical composition comprising thecomposition of any one of claim 60 or 64, and a pharmaceuticallyacceptable excipient.
 67. A method for treating a subject having adisorder in which TNFα is detrimental, comprising administering to thesubject the composition of claim 66, thereby treating the subject havinga disorder in which TNFα is detrimental.
 68. The method of claim 67,wherein the disorder in which TNFα is detrimental is selected from thegroup consisting of: sepsis (including septic shock, endotoxic shock,gram negative sepsis and toxic shock syndrome), autoimmune diseases(including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritisand gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes,autoimmune uveitis, nephrotic syndrome, multisystem autoimmune diseases,lupus (including systemic lupus, lupus nephritis and lupus cerebritis),Crohn's disease and autoimmune hearing loss), infectious diseases(including malaria, meningitis, acquired immune deficiency syndrome(AIDS), influenza and cachexia secondary to infection), allograftrejection and graft versus host disease, malignancy, pulmonary disorders(including adult respiratory distress syndrome (ARDS), shock lung,chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonaryfibrosis, silicosis, idiopathic interstitial lung disease and chronicobstructive airway disorders (COPD), such as asthma), intestinaldisorders (including inflammatory bowel disorders, idiopathicinflammatory bowel disease, Crohn's disease and Crohn's disease-relateddisorders (including fistulas in the bladder, vagina and skin; bowelobstructions; abscesses; nutritional deficiencies; complications fromcorticosteroid use; inflammation of the joints; erythem nodosum;pyoderma gangrenosum; lesions of the eye, Crohn's related arthralgias,fistulizing Crohn's indeterminant colitis and pouchitis), cardiacdisorders (including ischemia of the heart, heart insufficiency,restenosis, congestive heart failure, coronary artery disease, anginapectoris, myocardial infarction, cardiovascular tissue damage caused bycardiac arrest, cardiovascular tissue damage caused by cardiac bypass,cardiogenic shock and hypertension, atherosclerosis, cardiomyopathy,coronary artery spasm, coronary artery disease, valvular disease,arrhythmias and cardiomyopathies), spondyloarthropathies (includingankylosing spondylitis, psoriatic arthritis/spondylitis, enteropathicarthritis, reactive arthritis or Reiter's syndrome and undifferentiatedspondyloarthropathies), metabolic disorders (including obesity anddiabetes, including type 1 diabetes mellitus, type 2 diabetes mellitus,diabetic neuropathy, peripheral neuropathy, diabetic retinopathy,diabetic ulcerations, retinopathy ulcerations and diabeticmacrovasculopathy), anemia, pain (including acute and chronic pains,such as neuropathic pain and post-operative pain, chronic lower backpain, cluster headaches, herpes neuralgia, phantom limb pain, centralpain, dental pain, opioid-resistant pain, visceral pain, surgical pain,bone injury pain, pain during labor and delivery, pain resulting fromburns, including sunburn, post partum pain, migraine, angina pain andgenitourinary tract-related pain including cystitis), hepatic disorders(including hepatitis, alcoholic hepatitis, viral hepatitis, alcoholiccirrhosis, al antitypsin deficiency, autoimmune cirrhosis, cryptogeniccirrhosis, fulminant hepatitis, hepatitis B and C and steatohepatitis,cystic fibrosis, primary biliary cirrhosis, sclerosing cholangitis andbiliary obstruction), skin and nail disorders (including psoriasis(including chronic plaque psoriasis, guttate psoriasis, inversepsoriasis, pustular psoriasis and other psoriasis disorders), pemphigusvulgaris, scleroderma, atopic dermatitis (eczema), sarcoidosis, erythemanodosum, hidradenitis suppurative, lichen planus, Sweet's syndrome,scleroderma and vitiligo), vasculitides (including Behcet's disease) andother disorders, such as juvenile rheumatoid arthritis (JRA),endometriosis, prostatitis, choroidal neovascularization, sciatica,Sjogren's syndrome, uveitis, wet macular degeneration, osteoporosis andosteoarthritis.